Characterization of a thermotolerant ROK-type mannofructokinase from Streptococcus mitis: application to the synthesis of phosphorylated sugars

Article abstract of DOI:10.1007/s00253-018-9018-1

Most of the “repressor, open reading frame, kinase” (ROK) proteins already characterized so far, and exhibiting a kinase activity, take restrictedly d-glucose as substrate. By exploring the sequenced bacterial diversity, 61 ATP-dependent kinases belonging to the ROK family have been identified and experimentally assayed for the phosphorylation of hexoses. These kinases were mainly found to be thermotolerant and highly active toward d-mannose and d-fructose with notable activities toward d-tagatose. Among them, the ATP-dependent kinase from the mesophile Streptococcus mitis (named ScrK_mitis) was biochemically characterized and its substrate spectrum further studied. This enzyme possessed impressive catalytic efficiencies toward d-mannose and d-fructose of 1.5 10⁶?s^?1?M^?1 and 2.7 10⁵?s^?1?M^?1, respectively, but also significant ones toward d-tagatose (3.5 10²?s^?1?M^?1) and the unnatural monosaccharides d-altrose (1.1 10⁴?s^?1?M^?1) and d-talose (3.4 10²?s^?1?M^?1). Specific activities measured for all hexoses showed a high stereopreference for d- over l-series. As proof of concept, 8 hexoses were phosphorylated in moderate to good yields, some of them described for the first time like l-sorbose-5-phosphate unusually phosphorylated in position 5. Its thermotolerance, its wide pH tolerance (from 7 to 10), and temperature range (> 85% activity between 40 and 70?°C) open the way to applications in the enzymatic synthesis of monophosphorylated hexoses.

Full text of DOI:10.1007/s00253-018-9018-1

Applied Microbiology and Biotechnology
https://doi.org/10.1007/s00253-018-9018-1
BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Characterization of a thermotolerant ROK-type mannofructokinase
from Streptococcus mitis: application
to the synthesis of phosphorylated sugars
Carine Vergne-Vaxelaire1 & Aline Mariage1 & Jean-Louis Petit1 & Aurélie Fossey-Jouenne1
&
Christine Guérard-Hélaine2,3 & Ekaterina Darii1 & Adrien Debard1 & Stessy Nepert1 & Virginie Pellouin1
&
Marielle Lemaire2,3 & Anne Zaparucha1 & Marcel Salanoubat1 & Véronique de Berardinis1
Received: 2 February 2018 /Revised: 29 March 2018 /Accepted: 10 April 2018
#
Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract
Most of the Brepressor, open reading frame, kinase^ (ROK) proteins already characterized so far, and exhibiting a kinase activity,
take restrictedly D-glucose as substrate. By exploring the sequenced bacterial diversity, 61 ATP-dependent kinases belonging to
the ROK family have been identified and experimentally assayed for the phosphorylation of hexoses. These kinases were mainly
found to be thermotolerant and highly active toward D-mannose and D-fructose with notable activities toward D-tagatose. Among
them, the ATP-dependent kinase from the mesophile Streptococcus mitis (named ScrKmitis) was biochemically characterized and
its substrate spectrum further studied. This enzyme possessed impressive catalytic efficiencies toward D-mannose and D-fructose
of 1.5 106 s−1 M−1 and 2.7 105 s−1 M−1, respectively, but also significant ones toward D-tagatose (3.5 102 s−1 M−1) and the
unnatural monosaccharides D-altrose (1.1104 s−1 M−1) and D-talose (3.4 102 s−1 M−1). Specific activities measured for all hexoses
showed a high stereopreference for D- over L-series. As proof of concept, 8 hexoses were phosphorylated in moderate to good
yields, some of them described for the first time like L-sorbose-5-phosphate unusually phosphorylated in position 5. Its thermo-
tolerance, its wide pH tolerance (from 7 to 10), and temperature range (> 85% activity between 40 and 70 °C) open the way to
applications in the enzymatic synthesis of monophosphorylated hexoses.
.
.
.
.
.
Keywords Hexokinase Fructokinase Thermotolerance ROK family Phosphorylated sugars Promiscuity
Introduction
but also as a mean of uptake. This reaction is catalyzed by
sugar kinases such as glucokinases (EC 2.7.1.2), fructokinases
(EC 2.7.1.4), and hexokinases (EC 2.7.1.1). While hexoki-
nases, mostly reported among eukaryotes, exhibit broad sub-
strate range tolerance, other sugar kinases have narrow sugar
substrate specificity. Many bacterial sugar kinases are grouped
into the ROK family (repressor, open reading frame, kinase), a
large family of sugar metabolism-related proteins (Conejo
et al. 2010; Larion et al. 2007; Titgemeyer et al. 1994).
Enzymatic syntheses of mono O-phosphorylated sugars
have been described in the literature (Wohlgemuth et al.
2017) especially by transketolase- or aldolase-catalyzed C-C
bond formation or by selective phosphorylation catalyzed by
kinases (Charmantray et al. 2009; Guérard-Hélaine et al.
2014; Hélaine et al. 2015; Sánchez-Moreno et al. 2012;
Shaeri et al. 2008; Wohlgemuth et al. 2017). Chiral phosphor-
ylated sugars are of importance as central metabolites in the
non-oxidative pentose phosphate pathway, in the Calvin cy-
cle, and of particular interest for the study of various metabolic
Phosphorylation of monosaccharides is a highly important
stage of carbohydrate metabolism in prokaryotes, mainly for
preparing carbohydrates for degradation and transfer reactions
Carine Vergne-Vaxelaire and Aline Mariage contributed equally to this
work.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00253-018-9018-1) contains supplementary
material, which is available to authorized users.
* Véronique de Berardinis
vberard@genoscope.cns.fr
1
Génomique métabolique, Genoscope, Institut François Jacob, CEA,
CNRS, Univ Evry, Université Paris-Saclay, 91057 Evry, France
2
Clermont Université, Université Blaise Pascal, ICCF, BP 10448,
63000 Clermont-Ferrand, France
3
CNRS, UMR 6296, BP 8002663177, Aubière, France

Appl Microbiol Biotechnol
diseases such as diabetes, cancer, immunodeficiency, athero-
sclerosis, mucoviscidosis (Desvergnes et al. 2012), as well as
unintended virus or microorganism developments (Shaeri
et al. 2008, Wohlgemuth 2009, Solovjeva and Kochetov
2008, Schorken 1998). Given the vital roles of such com-
pounds and, more particularly, of phosphorylated hexoses
for metabolism studies but also organic chemistry, new ki-
nases allowing their straightforward synthesis are needed.
Among established strategies to provide enzymes possessing
new or improved properties, exploring protein natural diver-
sity is one of the most efficient (Bastard et al. 2014; Davids
et al. 2013; de Berardinis et al. 2017; Guérard-Hélaine et al.
2015; Vergne-Vaxelaire et al. 2013; Zhao et al. 2013). In this
study, we explored the sequenced bacterial diversity to find
novel ATP-dependent C6 sugar kinases able to phosphorylate
unusual various substrates such as D-tagatose, D-psicose and
L-sorbose, rare or unnatural hexoses. Among the 61 ATP-
dependent kinases found to be active toward various hexoses,
were from Agilent technologies (Santa Clara, USA).
Enzymatic spectrophotometric screenings in microplates were
performed on a SpectraMax Plus384 (Molecular Devices,
Sunnyvale, USA). Spectrophometric assays of ScrKmitis were
recorded on a Safas UVMC2 (Safas, Monaco) thermostated
when specified with a refrigerated/heating circulator Corio
CD-200F (Jubalo®, Seelbach, Germany) using microcells
high-precision cell quartz with 10-mm light path (Hellma
Analytics, Müllheim, Germany). Cell crude extracts in 96-
microwell plates were heated using a tested thermocycler
GeneAmpTM PCR system 9700 (Applied Biosystem, Foster
City, USA). NMR spectra were recorded on a Bruker (Bruker,
Billerica, USA) 300 or 600 MHz spectrometer (Evry
1
University, France) for H and 13C experiments, and on a
1
Bruker 500 MHz (ICSN, France) for H-31P experiments.
Chemical shifts are expressed in ppm and referenced with
residual solvent signals. Coupling constant values (J) are giv-
en in hertz. NMR spectra of α form and β form are respec-
tively noted as α and β. UHPLC analyses were performed on
a UHPLC U3000 RS 1034 bar (Thermo Fisher Scientific,
Waltham, USA) equipped with diode array detector
DAD3000 and an evaporative light scattering detector
(ELSD) 6100 (Esa Chromachem ®). HPLC purifications
were done on a semi-preparative HPLC system promi-
nence LC-20AP (Shimadzu, Kyoto, Japan) equipped with
an ELSD-LTII (Shimadzu, Kyoto, Japan) and a fraction
collector FRC-10A (Shimadzu, Kyoto, Japan). LC-HRMS
analyses were performed on a Thermo Scientific™
Dionex™ UltiMate™ 3000 thermostated column compart-
ment rapid separation (TCC-3000RS) LC system (Thermo
Fisher Scientific, Waltham, USA) coupled to ultra-high
resolution Orbitrap Elite hybrid mass spectrometer
(Thermo Fisher Scientific, Waltham, USA) equipped with
electrospray ionization (ESI) source. Ion exchange column
purifications were performed on a Dowex® 1X8-200-400
mesh resin purchased from Acros Organics (Thermo Fisher
Scientific, Waltham, USA).
a thermotolerant kinase from Streptococcus mitis (ScrKmitis
)
was selected to be further studied and its biocatalytic capabil-
ities were characterized. Data regarding substrate promiscuity,
reaction conditions (pH and temperature), biochemical prop-
erties, as well as the structural identification of some phos-
phorylated compounds, were provided.
Materials and methods
Chemicals and materials
All chemical reagents were purchased from commercial
sources and used without additional purification. D-(+)- and
L-(−)-allose, D-(+) and L-(−)-altrose, L-(−)-mannose, D-(−)-
and L-(+)-gulose, D -(+)- and L-(−)-idose, L-(−)-galactose,
D-(+)- and L-(−)-talose, L-psicose, L-(+)-fructose, and L-(+)-
tagatose were purchased from Carbosynth (Compton, berk-
shire, UK). D-(−)-fructose, D-psicose, D-(+)- and L-(−)-
sorbose, D-(−)-tagatose, D-(+)- and L-(−)-glucose, D-(+)-man-
nose, D-(−)- and L-(+)-threose, D-xylulose, D-(−)-lyxose, D-(+
)-xylose, D-(−)- and L-(+)-arabinose, D-(−)-ribose, D-(−)- and
L-(+)-erythrose, D-(+)-galactose, L-(+)-erythrulose, D-(+)-cel-
lobiose and N-acetyl- D-(+)-glucosamine, adenosine triphos-
phate (ATP), phosphoenolpyruvic acid monopotassium salt
(PEP), β-Nicotinamide adenine dinucleotide reduced
(NADH) and β-Nicotinamide adenine dinucleotide (NAD),
isopropyl-β- D -thiogalactoside (IPTG), 2-(N-
morpholino)ethanesulfonic acid (MES), 2-Amino-2-(hy-
droxymethyl)-1,3-propanediol, enzymes lactate dehydroge-
nase (LD), and pyruvate kinase (PK) from rabbit muscle were
purchased from Sigma Aldrich (MilliporeSigma, StLouis,
USA). Bovine serum albumin was purchased from Bio-Rad.
Oligonucleotides were from Sigma-Aldrich (MilliporeSigma,
StLouis, USA). E. coli strains BL21-CodonPlus (DE3)-RIPL
Cloning, expression, and purification
Primers were chosen and genes were cloned with a histidine
tag in N-terminal part in a pET22b(+) (Novagen) modified for
ligation independent cloning as already described (Bastard
et al. 2014). All primers and strains of the 90 selected ROK-
type kinases are listed in Table S1 and named from KIN1 to
KIN90. All the strains along with their identifiers were pur-
chased from DSMZ, CIP, or ATCC collections. When DNA
samples corresponding to the gene encoding the selected en-
zyme was not available, PCR was performed on the DNA of
another strain from the same species as noted in Table S1. All
sequences were verified. In these cases, when the protein se-
quence or the strain is not identical to that of the selected
enzyme, the sequence was deposited to the European

Appl Microbiol Biotechnol
Nucleotide Archive (ENA) with ENA id listed in Table S1.
Assigned Uniprot id are A0A2K4X4D6, A0A2K4X4G8,
A0A2K4X4D1, A0A2K4X4E5, A0A2K4X4D2,
A0A2K4X4E4, A0A2K4X4I2, and A0A2K4X4C6. Each ex-
pression plasmid was transformed into Escherichia coli
BL21-CodonPlus (DE3)-RIPL. Cell culture, induction of pro-
tein production, and cell lysis were conducted as previously
published (Bastard et al. 2014). Enzymes were purified in 96
microwells using HisLink™ 96 protein purification system
(Promega). The elution buffer was 50 mM glycylglycine
(pH 7.5), 50 mM NaCl, 250 mM imidazole, and 10% glycerol.
Samples were analyzed by SDS-PAGE using the NuPAGE
system (Invitrogen), see Table S2. Large-scale purification
of ScrKmitis was conducted from a 400-mL culture as de-
scribed elsewhere (Kreimeyer et al. 2007). The storage
buffer was Tris pH 8, 50 mM, NaCl 50 mM, 1 mM DTT,
and glycerol 15%. Purified enzymes were stored at −
80 °C. The sample was analyzed by SDS-PAGE using the
NuPAGE system. Protein concentration was determined by
the Bradford method, with bovine serum albumin as the
standard (Bio-Rad). Size exclusion chromatography was
conducted on Superdex using a 200 10/300GL increase
column (GE Healthcare Life Sciences).
Phylogenetic analyses
A careful inventory of Bknown^ ROK-type sugar kinases was
done searching as exhaustive as possible fructo-, glucose-, and
hexo-kinases described in the literature and related to experi-
mental data. Using Swissprot database information’s, only pro-
teins sharing the Interpro signature IPR000600 specific of ROK
family were selected and listed in Table S4 and S5. These ROK
kinases were aligned together with the 61 kinases of this study.
Pairwise sequence alignments were calculated with gapped
BLASTp and the BLOSUM62 scoring matrix. Phylogenetic
analysis was performed using MAFFT (Katoh et al. 2002) for
multiple sequence alignment, QUICKTREE (Howe et al. 2002)
for tree building, and iTOL (Letunic and Bork 2016) for tree
rendering. All the UniprotKB identifiers of the enzyme are re-
ported in the phylogenetic trees. Specific activities (Table S3) of
the 61 kinases were quantitatively symbolized and reported in
the phylogenetic tree. ROK-type sugar kinases from the litera-
ture have been included and the activities described in their
corresponding article (Table S4 and Table S5) were qualitatively
reported in the phylogenetic tree. In addition, 3 known ROK-
type repressors (XylR from Bacillus subtilis (Kreuzer et al.
1989) and Staphylococcus xylosus (Sizemore et al. 1991) and
NagC from E. coli (Plumbridge 2001) were added. Respective
Uniprot id were P16557, P27159, and P0AF20.
Enzymatic screening assay
Enzymes thermotolerance
General screenings conducted in 384-microwell plates were
done by spectrophotometry analysis (SpectraMax Plus384
(Molecular Devices, Sunnyvale, USA)) at 25 °C in a final
volume of 70 μL. ATP-dependent activities for phosphoryla-
tion were determined by monitoring the consumption of the
NADH spectrophotometrically at A340 using the coupled as-
say (Jaworek and Welsch 1985) employing pyruvate kinase
(PK) and lactate dehydrogenase (LD) according to Beer-
Lambert’s law and molar absorptivity of β-NADH (ɛ =
6220 M−1 cm−1). The assay mixture comprised 30 mM sub-
strate, 0.2 mM NADH, 2.5 mM ATP, 5 mM MgCl2, 5 mM
PEP, 1 U PK, 2.3 U LD, and 50 mM glycylglycine buffer pH
7.5. The reaction was initiated by addition of 10 μL of purified
enzyme (average of 500 μg/mL). The background noise was
estimated and removed for each enzyme with a supplemental
microplate similarly treated but with a mixture lacking hexose
substrate.
Volumes of 200 μL of cell crude extracts in 96 microwells
were incubated at 70 °C for 20 min in thermocycler. The
microplates were centrifuged at 4000 rpm for 20 min in a
centrifuge 5810R (Eppendorf). Supernatants were transferred
in a new microplate, analyzed by SDS-PAGE using the E-
PAGE High-Throughput Gel system (Invitrogen), and specific
activity measured. Protein concentrations were determined by
the Bradford method, with bovine serum albumin as standard
(Bio-Rad). ATP-dependent activities for phosphorylation of
D-fructose, D-tagatose, and D-glucose were determined by
monitoring the consumption of the NADH spectrophotomet-
rically as described for the enzymatic screening assay.
Characterization of ScrKmitis
All specific activities and kinetic parameters were determined
from duplicate experiments by monitoring the consumption of
the NADH spectrophotometrically as described for the enzy-
matic screening assay. All reactions (except when notified)
were performed on purified ScrKmitis at 25 °C in 100-μL scale
using microcuvettes.
ROK signatures analysis
Pairwise sequence alignments were performed using Clustal
Omega website (https://www.ebi.ac.uk/Tools/msa/clustalo/).
The 61 ROK-type kinases and the ROK-type sugar kinases
from the literature (Table S3, S4 and S5) were used for the
alignment analysis. ROK signatures were searched according
to Conejo’s work (Conejo et al. 2010).
Protocol A: The assay mixture (100 μL) comprised
50 mM hexose (except for D-fructose 2.5 mM), 0.2 mM
NADH, 2.5 mM ATP, 5 mM metal ion, 5 mM

Appl Microbiol Biotechnol
phosphoenolpyruvate (PEP), 1 U PK, 2.3 U LD, and
50 mM glycylglycine buffer pH 7.5. The initial slope
measured at 340 nm for 10 min after addition of purified
ScrKmitis determined the specific activity of the enzyme.
Protocol B: Protocol carried out in end-point to ensure the
independence of the pyruvate kinase and lactate dehydro-
genase assay system previously validated between pH 6.5
and 9 (data not shown). Assay mixture was composed
of 2.5 mM D-fructose, 2.5 mM neutralized ATP, 5 mM
MgCl2, and 50 mM specified buffer at specified pH.
To 100 μL of the assay mixture preincubated 3 min at
the specified temperature was added purified ScrKmitis
and the reaction was stirred at 400 rpm for 2 min at the
specified temperature. After acidic quench to pH = 1
(HCl 4–10 M), neutralization to pH 6.5–9 (KOH 4–
10 M), the ADP formed was assayed by pyruvate ki-
nase and lactate dehydrogenase system (0.2 mM
NADH, 1 U PK, 5 mM neutralized PEP, 2.3 U LD)
by measuring the difference in absorbance at 340 nm
after total consummation of ADP.
(vi) Determination of kinetic parameters of ScrKmitis.
Kinetic constants were determined on purified
ScrKmitis by using the spectrophotometric coupled as-
say employing pyruvate kinase and lactate dehydroge-
nase system. Assay mixture was composed of 0.25 mM
NADH, 2.5 mM ATP, 5 mM MgCl2, 5 mM PEP, 1 U
PK, 2.3 U LD, and 50 mM glycylglycine buffer pH 7.5.
The kinetic constants for ATP were determined with
0.25 mM NADH, 10 mM D-fructose, 5 mM MgCl2,
5 mM PEP, 1 U PK, 2.3 U LD, and 50 mM
glycylglycine buffer pH 7.5. The following substrate
concentrations were used: ATP from 0.005 to 2.5 mM,
D-fructose from 0.1 to 10 mM, D-altrose from 1 to
100 mM, D-mannose from 0.01 to 20 mM, D-glucose
from 5 to 400 mM, L-sorbose from 5 to 500 mM, D-
sorbose from 10 to 1200 mM. 100 μL of the assay
mixture was preincubated until the complete consump-
tion of ADP present in commercial ATP, followed by
addition of ScrKmitis. Kinetic parameters were deter-
mined by fitting data by non-linear analysis of initial
rates using SigmaPlot version 9.0 (Systat Software).
(vii) ATPase activity of ScrKmitis. The ATPase activity was
determined by using the spectrophotometric coupled
assay employing pyruvate kinase and lactate dehydro-
genase system. Assay mixture was 0.25 mM NADH,
5 mM MgCl2, 5 mM PEP, 1 U PK, 2.3 U LD and
50 mM glycylglycine buffer pH 7.5, and 2.5 mM
ATP. 100 μL of the assay mixture was preincubated
until the complete consumption of ADP present in com-
(i) Specific activities of ScrKmitis toward various substrates.
Specific activities of ScrKmitis were determined by using
protocol A with MgCl2 as metal ion. Tested substrates
were (D- and L-form) allose, altrose, glucose, mannose,
gulose, idose, galactose, talose, psicose, fructose, sorbose,
tagatose, arabinose, erythrose, and threose. In addition, D-
ribose, D-xylose, D-lyxose, D-xylulose, L-erythrulose, D-
cellobiose, and N-acetyl-D-glucosamine were also assayed.
(ii) Thermotolerance of ScrKmitis. Thermotolerance of
ScrKmitis was confirmed with a cell crude extract obtain-
ed as already described (Kreimeyer et al. 2007) from
100 mL of culture. A volume of 8 mL of cell crude
extract was incubated 20 min at 70 °C and centrifuged
at 8000 rpm 30 min. The clear cell crude extract was
analyzed by SDS-PAGE using the Nu-PAGE system
(Invitrogen). Protein concentrations were determined
by the Bradford method, with bovine serum albumin
as standard (Bio-Rad). Specific activity of ScrKmitis
was measured with protocol A for D-fructose, D-man-
nose, and D-glucose.
mercial ATP, followed by addition of ScrKmitis
.
Determination of inorganic phosphate in the enzymatic
reaction in presence and absence of D-fructose was
followed with the colorimetric assay with molybdate.
Assay mixture was composed of 50 mM glycylglycine
buffer pH 7.5, 5 mM MgCl2, 12 μg of purified
ScrKmitis, and 2.5 mM of ATP in a final volume of
200 μl. In the reaction with hexose, 2.5 mM of D-
fructose was added. Control was done without adding
ScrKmitis. Aliquot of 50 μl was withdrawn at 0, 15, and
30 min and added to 150 μl of buffer (50% acetone,
25% sulfuric acid 5 M, and 25% ammonium molybdate
10 mM) and 10 μl of 1 M citric acid. The absorbance
was immediately measured at 355 nm.
(iii) Specific activity of ScrKmitis versus pH/buffer solution.
The protocol B was used at room temperature with the
following buffers: MES buffer (pH 4.5–6.8), TRIS HCl
buffer (pH 6.8–9.6), glycylglycine NaOH buffer (pH
7.0–12.4), TRIS NaOH buffer (pH 11.0–12.4).
(iv) Specific activity of ScrKmitis versus temperature. The
protocol B was used in glycylglycine buffer pH 7.5 at
15, 20, 30, 40, 45, 50, 55, 60, 65, 70, and 80 °C.
(v) Specific activity of ScrKmitis versus metal ions: the pro-
tocol A was used with 2.5 mM of D-fructose. Tested
inorganic salt at 5 mM were MgCl2, MnCl2, CoCl2,
NiCl2, ZnCl2, CuCl2, or CaCl2.
HPLC conditions
HPLC-ELSD analyses were conducted on a Zic-pHilic
(Merck, Darmstadt, Germany) column (4.6 × 150 mm;
5 μm) with the following conditions: mixture of NH4HCO2
20 mM pH 3.6/MeCN as eluent with a linear gradient (ratio
20/80 to 60/40 in 15 min, then 60/40 during 5 min); flow

Appl Microbiol Biotechnol
0.5 mL min−1; temperature 30 °C; injection volume 10 μL.
ELSD parameters Teva = 70 °C; Tneb = 40 °C; attenuation = 5.
Semi-preparative HPLC purifications were conducted
on a Zic-Hilic (Merck, Darmstadt, Germany) column
(250 × 10 mm; 5 μm) with the following conditions: mix-
ture of NH4AcO 10 mM pH 7.7/MeCN as eluent with a
linear gradient (ratio 20/80 to 60/40 in 15 min, then 60/40
during 5 min); flow 4 mL min−1. ELSD parameters: T =
70 °C; gain = 5: filter = 1 s.
LC-HRMS analyses were performed on a Zic-pHilic
(Merck, Darmstadt, Germany) column (4.6 × 150 mm;
5 μm) with the following chromatographic conditions: mix-
ture of (NH4)2CO3 10 mM pH 9.9 (adjusted with NH4OH)/
MeCN as eluent with a linear gradient (20/80 for 2 min, 20/80
to 60/40 in 20 min, then 60/40 during 8 min); flow
0.5 mL min−1; temperature 40 °C; injection volume 10 μL.
The Orbitrap Elite mass spectrometer was operated in the ESI
negative ion mode. For LC-MS and MS/MS (collision in-
duces dissociation, CID) experiments, the resolving power
was set at 60000 m/Δm (full width at half maximum,
FWHM at m/z = 400). Ion spray (IS) was set at − 4.2 kV and
capillary temperature at 275 °C. Sheath gas and auxiliary gas
flow rates were set at 60 and 50 arbitrary units (a. u.), respec-
tively. Mass spectra were acquired over an m/z range from m/z
50 up to m/z 1000. Raw data were analyzed using the Qual-
browser module of Xcalibur version 2.2 (Thermo Fisher
Scientific, Waltham, USA).
Purification by ion exchange chromatography The freeze-
dried product was dissolved in NH4HCO3 buffer 0.012 M
and purified by ion exchange chromatography using a
−
Dowex (1X8-200-400 mesh, HCO3 ) resin with a non-linear
gradient of various NH4HCO3 concentrations: 0.012 M,
0.03 M, 0.06 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, and 0.6 M (2
vol columns). Fractions containing the compound (HPLC-
ELSD analyses 50 μL of eluting fractions +50 μL of HPLC
eluent NH4HCO2 20 mM pH 3.6/MeCN 8/2) were combined
and freeze-dried to afford the phosphorylated hexose. Three
successive freeze-drying (solid recovery, dissolution in water)
were necessary to ensure the complete removal of buffer.
Purification by semi-preparative HPLC The freeze-dried prod-
uct was dissolved in HPLC eluent NH4AcO 10 mM pH 7.7/
MeCN 8/2) and injected on Zic-Hilic semi-preparative col-
umn (injection volumes 2–2.5 mL). Positive fractions
(ELSD detection) were combined and freeze-dried to afford
the phosphorylated hexose. Three successive freeze-drying
(solid recovery, dissolution in water) were necessary to ensure
the complete removal of buffer.
D-(+)-altrose-6P: The reaction was conducted under condi-
tions A with 0.53 mg of ScrKmitis in 10 h and the product
purified by ion-exchange chromatography. Isolated yield:
57% (25 mg of white solid). NMR 13C was identical to the
one found in the literature (Franke et al. 1985). HRMS ESI−,
m/z calcd. for [C6H12O9P]− = 259.02244; found 259.02214.
D-(+)-glucose-6P: The reaction was conducted under con-
ditions A with 0.38 mg of ScrKmitis in 8 h and the product
purified by ion-exchange chromatography. Isolated yield:
56% (22 mg of white solid). Present forms: α-pyranose
Biocatalytic synthesis of phosphorylated hexoses
1
Conditions A In a Greiner tube (15 mL) with a screw cap was
poured 7.5 mL of a media containing 50 mM of TRIS HCl
buffer (375 μL of a 1 M solution at pH 8.1), 20 mM of hexose
(150 μmol, 1500 μL of a 100 mM solution), 0.5 mM of ATP
(3.75 μmol, 37.5 μL of a 100 mM solution at pH 6), 5 mM of
MgCl2 (37.5 μmol, 375 μL of a 100 mM solution), 25 mM of
PEP (187.5 μmol, 750 μL of a 250 mM solution at pH 6),
(35%) and β-pyranose (65%). H NMR (300 MHz, D2O) δ
5.12 (d, J = 3.63 Hz, H1α),4.54 (d, J = 7.26 Hz, H1β), 4.00–
3.85 (m, H6, H2, H4), 3.85–3.76 (m, H5), 3.62 (m, H3), 3.48
(m, H6), 3.45–3.35 (m, H3, H4), 3.61 (dd, J = 9.21 and
7.99 Hz); 13C NMR (75 MHz, D2O) δ 96.3 (C1β), 92.4
(C1α), 75.6 (C3α), 75.4 (d, J = 7.55 Hz, C5β), 74.4 (C2β),
72.6 (C3α),71.8 (C2α), 71.1 (d, J = 7.71 Hz, C5α), 69.3 (C4),
63.4 (d, J = 4.48 Hz, C6 α and β) (Fig. S5). HRMS ESI−, m/z
calcd. for [C6H12O9P]− = 259.02244; found 259.02192.
D-(+)-mannose-6P: The reaction was conducted under con-
ditions A with 0.04 mg of ScrKmitis in 15 h and the product
purified by ion-exchange chromatography. For practical rea-
son, the reaction was carried out overnight but the conversion
was > 95% after 2 h 30 on 100 μL scale. Isolated yield: 77%
(30 mg of white solid). Present forms: α-pyranose (66%) and
15 U of PK, and the specified quantity of purified ScrKmitis
.
The reaction mixture was stirred at 400 rpm at 30 °C for the
indicated period of time then lyophilized overnight.
Conditions B In a Greiner tube (50 mL) with a screw cap
was poured 15 mL of a media containing 50 mM of TRIS
HCl buffer (750 μL of a 1 M solution at pH 8.1), 10 mM of
hexose (150 μmol, 1500 μL of a 100 mM solution), 10 mM
of ATP (150 μmol, 1500 μL of a 100 mM solution at pH 6),
5 mM of MgCl2 (75 μmol, 75 μL of a 1 M solution),
25 mM of PEP (375 μmol, 1500 μL of a 250 mM solution
at pH 6), 150 U of PK, and the specified quantity of purified
ScrKmitis. The reaction mixture was stirred at 400 rpm at
30 °C for the indicated period of time then freeze-dried
overnight.
1
β-pyranose (34%). H NMR (300 MHz, D2O) δ 5.03 (s,
H1α), under H2O (H1β), 3.93–3.85 (m, H6), 3.79–3.74 (m,
H2), 3.74–3.62 (m, H3α, H4α, H5α), 3.62–3.49 (m, H4β,
H3β), 3.28 (m, H5β);13C NMR (75 MHz, D2O) δ 94.4
(C1α), 94.1 (C1β), 75.6 (d, J = 7.69 Hz, C5β), 72.8 (C3β),
72.0 (d, J = 7.36 Hz, C5α), 71.5 (C2β), 70.9 (C2α), 70.1
(C3α), 66.5 (C4α), 66.2 (C4β), 63.1 (d, J = 4.34 Hz,

Appl Microbiol Biotechnol
C6αβ) (Fig. S6). HRMS ESI−, m/z calcd. for [C6H12O9P]− =
259.02244; found 259.02194.
(20 mg of white solid). Present forms: α-furanose (25%) and
β-furanose (75%). 1H NMR (300 MHz, D2O) δ 4.37–4.30 (m,
H4αβ), 4.20 (m, H3αβ), 4.12–3.82 (m, H5αβ, H6αβ), 3.61
(d, J = 4.29 Hz, H1α), 3.51 (d, J = 3.99 Hz, H1β) (Fig. S10);
13C NMR was identical to the one found in the literature
(Koerner Jr et al. 1980). HRMS ESI-, m/z calcd. for
[C6H12O9P]- = 259.02244; found 259.02211.
D-(+)-talose-6P: The reaction was conducted under condi-
tions Awith 0.53 mg of ScrKmitis in 7 h and the product purified
by ion-exchange chromatography. Isolated yield: 77% (30 mg
of white solid). Present forms: α-pyranose (41%), β-pyranose
1
(36%), α-furanose (13%) and β-furanose (10%). H NMR
(600 MHz, D2O) δ 5.27 (d, J = 4.41 Hz, H1’β),5.17 (d,
J = 1.62 Hz, H1α), 5.14 (m, H1’α), 4.73 (d, J = 1.08 Hz,
H1β), 4.36 (m, H3’β), 4.27 (m, H3’α), 4.10 (m, H5’α, H5α),
4.01–4.05 (m, H4’β, H2’β), 3.91 (m, H2’α, H3α), 3.87 (m,
H4’α, H6αβ), 3.82–3.84 (m, H4αβ, H6’β), 3.78 (m, H5’β,
H2β, H6’α), 3.73 (m, H2α), 3.70 (m, H3β), 3.65 (m, H5β);
13C NMR (150 MHz, D2O) δ 103.0 (C1’α), 96.4 (C1’β), 94.7
(C1β), 94.1 (C1α), 82.6 (C4’β), 82.1 (C4’α), 75.2 (C2’α), 74.3
(d, J = 7.67 Hz, C5β), 71.5 (C2β), 71.0 (d, J = 7.12 Hz, C5’α),
70.9 (C3’β), 70.8 (C2’β), 70.6 (C2α), 70.4 (C3’α), 70.1 (d,
J = 7.30 Hz, C5’β), 70.0 (d, J = 7.64 Hz, C5α), 69.0 (C3α),
68.2 (C3α), 68.0 (C4β), 65.4 (d, J = 4.99 Hz, C6’β), 65.3 (d,
J = 4.95 Hz, C6’α), 64.9 (C4α), 63.4 (d, J = 4.38 Hz, C6α),
63.1 (d, J = 4.30 Hz, C6β) (Fig. S7). HRMS ESI−, m/z calcd.
for [C6H12O9P]− = 259.02244; found 259.02211.
Results
Screening of a kinase collection
A kinase collection was built by a sequence driven approach
(Vergne-Vaxelaire et al. 2013) using the fructokinase FrK
from Zymomonas mobilis (Weisser et al. 1996; Zembrzuski
et al. 1992) as reference. Frk is the unique ROK-type
fructokinase (Uniprot ID: Q03417) with enzymatic character-
ization done on purified enzyme (Table S4) showing moderate
activity toward D-mannose in addition to D-fructose. Since
little is known about the substrate scope of ROK-type
fructokinase enzymes, thus, exploration of their substrate
specificity toward sugars is of great interest.
D-psicose-6P: The reaction was conducted under condi-
tions B with 3 mg of ScrKmitis in 4 days and the product
purified by ion-exchange chromatography. Isolated yield:
51% (20 mg of white solid). Present forms: α-furanose
A collection of 90 proteins representative of the diver-
sity among bacteria was selected from the UniProt data-
base. From these candidates, 65 corresponding genes were
successfully cloned with a poly-histidine tag in an expres-
sion vector (Table S1 and Table S2). After overexpression
of recombinant genes in Escherichia coli strain BL21,
cells were lyzed and proteins purified by nickel affinity
chromatography in 96 microwells (Fig. S1). The 63 suc-
cessfully purified enzymes were then screened against 4
ketohexoses (D-fructose, D-tagatose, D-sorbose, and D-
psicose) and 3 aldohexoses (D-glucose, D-mannose, and
D-galactose). The kinase activity of each candidate protein
was assessed in 384-microplates by spectrophotometrical-
ly monitoring at 340 nm, the consumption of the NADH
using the coupled generic assay employing pyruvate ki-
nase, and lactate dehydrogenase (Scheme S1). Sixty-one
enzymes very diverse in term of protein sequence (Fig.
S11) were found to catalyze the formation of at least
one phosphorylated hexose. In our conditions, no activity
could be detected toward D-galactose. Two enzymes
weakly purified showed no activity against any hexose
(no activity could be detected for C5 and C4 monosac-
charides too, data not shown). These kinases could be
classified in two groups according to their substrate spec-
ificity: a small group, gathering proteins active toward D-
glucose and a group, bringing together most of the pro-
teins (55 out of 61), exhibiting, at least, activities toward
both D-mannose and D-fructose (Fig. 1a). Noteworthy
among them, 42 kinases exhibited such a high activity
(> 50 U/mg) toward D-mannose and D-fructose that it
1
(35%) and β-furanose (65%). H NMR (300 MHz, D2O) δ
4.37–4.29 (m, H4αβ), 4.19–4.04 (m, H3αβ), 4.00–3.66 (m,
H5αβ, H6αβ), 3.55–3.45 (m, H1αβ) (Fig. S8); NMR 13C
was identical to the one found in the literature (Koerner Jr
et al. 1980). HRMS ESI−, m/z calcd. for [C6H12O9P]− =
259.02244; found 259.02206.
D-fructose-6P: The reaction was conducted under condi-
tions A with 0.04 mg of ScrKmitis in 30 min and the product
purified by semi-preparative HPLC. Isolated yield: 72%
(28 mg of white solid). Present forms: α-furanose (25%) and
β-furanose (75%); 13C NMR was identical to those found in
the literature (Sánchez-Moreno et al. 2012).
L-sorbose-5P: The reaction was conducted under condi-
tions A with 1.5 mg of ScrKmitis in 24 h and the product
purified by semi-preparative HPLC. Isolated yield: 67%
(26 mg of white solid). Present form: α-pyranose (>
1
95%). H NMR (500 MHz, D2O) δ 3.82 (m, H5), 3.71
(dd, J = 11.08 and 5.38 Hz, H6a), 3.62 (t, J = 9.15 Hz,
H4), 3.55–3.49 (m, H1a, H6b), 3.39 (d, J = 9.50 Hz, H3),
3.36 (d, J = 11.75 Hz, H1b); 13C NMR (125 MHz, D2O) δ
97.5 (C2), 73.5 (d, J = 3.09 Hz,C4), 72.4 (d, J = 4.40 Hz,
C5), 70.4 (C3), 63.6 (C1), 60.9 (d, J = 2.93 Hz, C6) (Fig.
S9). HRMS ESI-, m/z calcd. for [C6H12O9P]- =
259.02244; found 259.02208.
D-tagatose-6P: The reaction was conducted under condi-
tions A with 0.38 mg of ScrKmitis in 2 h and the product
purified by semi-preparative HPLC. Isolated yield: 51%

Appl Microbiol Biotechnol
M
C N
STPG
F
C
A
B
Fruktokinases
Repressors
Fig. 1 Phylogenetic tree with the 61 ROK-type kinases compared to
known ROK-type proteins. Activity of known ROK-type sugar kinases
from the inventory (Table S4 and Table S5) are noted with gene name put
in parenthesis next to Uniprot identifier. Their activities toward substrates
described in the corresponding publications have been symbolized qual-
itatively with a star and mostly have not been tested in this study.
Experimentally data from this study are symbolized by squares whose
size is categorized according to specific activities (Table S3 and
Table S6). Frk used as reference set for the sequence driven approach is
written in blue. The most promiscuous and efficient enzyme in our con-
ditions of screening is written in red. Dotted rectangle gathers known
ROK-type repressors whereas green rectangle gathers fructokinases. a
Activity toward D-fructose (F), L-sorbose (S), D-tagatose (T), D-psicose
(P), D-glucose (G), D-mannose (M). b Activity toward D-cellobiose (C)
and N-acetyl-D-glucosamine (N). Bootstrap values superior to 80 have
been noted on the branch of the phylogenetic tree
was not possible to determine their specific activity in the
generic screening conditions. Moreover, most of these ki-
nases were also active toward D-tagatose and some of
them had low activity toward L-sorbose or D-psicose
(Table S3). It may be noted that, in our conditions, most
of the fructo- and glucokinases were weakly active toward
D-glucose and D-fructose, respectively.
61 ROK-type sugar kinases
Since Frk from Z. mobilis used as reference protein belongs to
ROK-type family, we investigated the ROK conserved se-
quence features (Conejo et al. 2010) among the 61 kinases.
From a literature survey, a careful inventory of sugar kinases
was done. For each of these kinases, the conserved Interpro

Appl Microbiol Biotechnol
domain IPR000600 specific of ROK family was searched
allowing the identification of 29 ROK-type sugar kinases re-
lated to a publication but not always biochemically character-
ized. The resulting glucokinases, allokinases, ATP-
glucomannokinases, β-glucoside kinases, N-acetyl-D-glucos-
amine kinases, and fructokinases (listed in Table S4 and
Table S5) were added to the multiple sequence alignment of
the 61 kinases.
First, the two glycine-rich ROK consensus motifs corre-
sponding to ExGH and a Zn-binding motif (Conejo et al.
2010) were found in all experimentally tested kinases of this
study confirming their belonging to ROK family (Fig. S12).
Nevertheless, we observed that the ATP/Mg2+ binding site
(DXGXT), described as common to ROK-type kinases
(Holmes et al. 1993) was shared as expected by glucokinases,
allokinases, ATP-glucomannokinases, β-glucoside kinases,
and N-acetyl-D-glucosamine kinases but not by fructokinases
(Fig. S13). For this sub-family, glutamate is always substituted
by aspartate in their ATP/Mg2+ binding site (EXGXT)
confirming the predictions based on structural analysis
(Conejo et al. 2010). In addition, we also observed that the
Thr/Pro/Lys triad was conserved among the fructokinases and
absent within other ROK-type sugar kinases (Fig. S14)
partitioning clearly these families, corroborating the hypothe-
sis formulated by (Conejo et al. 2010).
To evaluate the distribution of these 61 sugar kinases
among the whole ROK family, our experimental data were
mapped on a phylogenetic tree (Fig. 1a) built with the 61
characterized kinases of this study supplemented with the
known ROK-type kinases (see the inventory in Table S4 and
Table S5) and ROK-type repressors (two XylR (Kreuzer et al.
1989; Sizemore et al. 1991) and NagC (Plumbridge 2001)).
Glucokinases, including the ones found in this study, are
grouped together with the ROK-type repressors, forming a
group significantly distinct to the one of the fructokinases.
Actually, the 4 known fructokinases (ScrK(Sato et al. 1993),
FrK (Scopes et al. 1985) GmuE (Nocek et al. 2011) and Mak
(Sproul et al. 2001)) and the experimentally proved
fructokinases are not significantly phylogenetically related to
other ROK-type sugar proteins. Notably, glucokinases are
found clusterized with kinases described to be β-glucoside
kinases BglK (Thompson et al. 1992) and N-acetyl-
glucosamine kinases NagK (Uehara and Park 2004). In order
to test the ability of the sugar kinase collection to phosphory-
late glucose derivatives, ATP-dependent activities for phos-
phorylation of D-cellobiose and N-acetyl-D-glucosamine were
assayed (Table S6 and Fig. 1b). The results showed that only
two glucokinases were active toward D-cellobiose whereas 5
glucokinases were active toward N-acetyl-D-glucosamine. In
our conditions, no fructokinase was found significantly active
toward these derivatives.
enzyme thermotolerance was assessed. Enzyme thermotoler-
ance was determined by incubation of cell crude extract in 96-
microplates at 70 °C for 20 min. Thirty-six percent of
expressed enzymes were significantly visible to the eye on
SDS gel, suggesting their thermotolerance (Fig. S2). ATP-
dependent activities for phosphorylation of D-fructose, D-
tagatose, and D-glucose were determined as previously
described (Fig. S4). Twenty-three kinases showed no signifi-
cant loss of activity (Fig. 1c and Table S7). From these
thermotolerant promiscuous kinases, one enzyme from
Streptococcus mitis (Uniprot id: E1M3M4) was selected for
its ability to phosphorylate most of the tested hexoses.
Purification and biochemical properties of E1M3M4
from Streptococcus mitis NCTC 12261 (DSM 12643)
E1M3M4 shares 72% of identity with the fructokinase ScrK
from Streptococcus mutants (ScrKmutants, Uniprot ID:
Q07211) involved in the sucrose transport through a
phosphoenolpyruvate-dependent phosphotransferase system
(PTS) (Sato et al. 1993). An apparent Vmax for D-fructose of
20.0 pmol/min/mg of protein was reported on cell crude ex-
tract by (Sato et al. 1993). In addition, the genes encoding for
E1M3M4 and ScrKmutants share a similar genomic context
(Fig. S15) including a predicted sucrose enzyme II (ScrA)
and sucrose-6P hydrolase (ScrB) (Sato et al. 1989) usually
involved in disaccharide uptake by phosphoenolpyruvate-
dependent phosphotransferase system (PTS system) (Postma
et al. 1993). Based on these features, E1M3M4 was named
ScrKmitis. Purified ScrKmitis was obtained by nickel affinity
chromatography purification (47.5 mg from 100 mL of cul-
ture). SDS gel showed a single band corresponding to a cal-
culated molecular mass of 32.8 kDa (Fig. S3). Size exclusion
chromatography indicated that ScrKmitis is a homodimer, as
previously reported for the fructokinase of Zymomonas
mobilis (FrKmobilis) (Scopes et al. 1985). Biochemical proper-
ties of this purified enzyme were as follows.
(i) Substrate specificity
To determine the substrate range of ScrKmitis, specific ac-
tivities were calculated for all hexoketoses and hexoaldoses
(Table 1) in the D- and L- series and some C4 and C5 mono-
saccharides. ScrKmitis showed an unprecedented substrate
scope with high activities toward D-fructose (287 U mg−1)
and D-mannose (395 U mg−1) (Table 1, entry 10 and 4).
Significant specific activities (> 0.1 U mg−1 of protein) were
measured for all natural hexoses except D-galactose. ScrKmitis
phosphorylated also rare sugars, like for D-psicose, D-allose,
and D-gulose, with low specific activities (< 0.1 U mg−1 of
protein) (Table 1, entries 9, 1, and 5 respectively). Unnatural
monosaccharides were also phosphorylated, D-altrose with
high specific activities (105 U mg−1, Table 1 entry 2) and D-
Guided by potential biotechnological applications of these
promiscuous 61 kinases for the synthesis of rare sugars,

Appl Microbiol Biotechnol
Table 1 Specific activities of purified ScrKmitis toward hexoses (D- and
L-series)
corresponding to the expected mass of ScrKmitis (Fig.
S3b) showing that heat treatment can be used as a quick
and easy purification method. Specific activities were then
measured for D-fructose, D-mannose, and D-glucose, and
compared to ones obtained with ScrKmitis purified by nick-
el affinity chromatography. Loss of activity was estimated
between 20 and 30% (Table S8).
Entry
Substrates
Specific activity
(U/mg of enzyme)
D
L
C6-Aldose substrates
1
2
3
4
5
6
7
8
Allose
0.1
0.4
Altrose
Glucose
Mannose
Gulose
Idose
105
4.8
1
(iii) pH and temperature activity profiles
nd
395
< 0.1
< 0.1
0
< 0.1
< 0.1
< 0.1
< 0.1
< 0.1
Several parameters influencing the enzymatic activity have
been tested. In order to define the optimal pH, specific activ-
ities toward D-fructose were determined using various buffer
and pH (Fig. 2). Buffers ranging from pH 4.5 to pH 12.4 were
used. ScrKmitis displayed a relatively broad pH profile with a
maximum activity at basic pH (7 to 10). Activities dropped at
pH values below 5.9 and above 11.0.
Unlike ROK glucokinases, no ROK fructokinase has
been shown to be active at high temperatures. Since
ScrKmitis from the mesophilic bacteria Streptococcus mitis
was shown to be thermotolerant, we investigated its tem-
perature activity profile (Fig. 3). Assays were carried out
toward D-fructose at various temperatures ranging from 15
to 80 °C. Its maximum activity was found at approximately
55 °C, leading to an about two-fold higher activity com-
pared to incubation at room temperature. The activity de-
creased rapidly at temperature higher than 70 °C but high
activity (> 85%) was maintained within a relatively wide
temperature range of 40–70 °C.
Galactose
Talose
4.7
c6-Ketose substrates
9
Psicose
< 0.1
287
0.4
0.24
< 0.1
0.65
0.15
10
11
12
Fructose
Sorbose
Tagatose
15
One unit (U) of enzyme activity was defined as the amount of enzyme
that catalyzed the conversion of 1 μM of substrate per minute
nd not detected
talose and D-idose with lower activities (4.7 and < 0.1 U mg−1
respectively, Table 1 entries 8 and 6). Some L-monosaccha-
rides were also phosphorylated such as L-sorbose and L-altrose
(Table 1, entries 11 and 2, respectively) even if the activity
was generally higher for D-configuration than L-configuration.
Moreover, some C4 and C5 monosaccharides revealed to be
also substrates, notably D-lyxose (0.16 U mg−1). Traces of
activity could be detected for D-erythrose (0.04 U mg−1) and
L-erythrulose (0.018 U mg−1). In our reaction conditions D-
ribose, D- and L-arabinose, D-xylose, D-xylulose, L-erythrose,
D- and L-threose, D-erythrulose were not substrates.
In addition, ScrKmitis showed a low ATPase activity in our
conditions (KM = 0.9 mM; kcat/KM = 32 s−1 M−1).
Nevertheless, in presence of fructose, no release of Pi was
detected using acidified ammonium molybdate assay
(Heinonen and Lahti 1981) (Fig. S16). To our knowledge, this
feature was only described for sugar kinases from yeast
(Rother et al. 2017; Rudolph and Fromm 1971).
(iv) Metal ion requirement
ScrKmitis metal ion activity profile (Fig. 4) conducted with
5 mM of cation and D-fructose as substrate indicated a maxi-
mal activity with Mg2+ and Co2+ (relative activity 100 and
98.5%, respectively). The activity remained important with
Mn2+ (79.3%) but dropped drastically in the presence of
Zn2+, Ca2+, or Ni2+ (24.1, 12.9, and 6.5%, respectively). No
activity was detected in presence of 5 mM of Cu2+ or in ab-
sence of metal cation.
Kinetic constants
(ii) Thermotolerance
In the course of our characterization study, kinetic constants
were determined for 8 hexoses for which specific activities
were found higher than 0.1 U mg−1 of enzyme (Table 1).
Lowest KM were measured for D-mannose and D-fructose with
respective values of 150 and 730 μM compared, for example,
to 23 mM for D-glucose (Table 2). ScrKmitis was highly effec-
tive toward D-mannose and D-fructose (kcat/KM = 1.5 106 and
2.7 105, respectively, Table 2, entries 2 and 3). An about 10-
fold lower catalytic efficiency was still recorded for the
To confirm the thermotolerance previously observed in
96-microwells, measurement of enzyme activity after heat
treatment was carried out. Heat treatment cell free extract
of ScrKmitis at 70 °C for 20 min was then done. Recovered
protein mass (42.0 mg from 100 mL of culture) was similar
to the one obtained by nickel affinity chromatography pu-
rification. SDS gel showed only one band of 32.8 kDa

Appl Microbiol Biotechnol
120,0
100,0
80,0
60,0
40,0
20,0
0,0
Fig. 2 Effect of pH on the
fructokinase activity of ScrKmitis
Error bars represent the standard
deviation of three independent
experiments
.
2,0
4,0
6,0
8,0
10,0
12,0
14,0
pH
MES
Gly-Gly-NaOH
TRIS-HCl
TRIS-NaOH
transformation of the isomer D-altrose (Table 2, entry 4) while
it was 1000-fold lower for D-tagatose, D-talose and D-glucose
(Table 2, entries 5–7).
of substrate in 30 min with only 0.005 mg/mL of enzyme. In
the absence of the regeneration system, the conversion
reached only 7% in 3 h with one equivalent of ATP or 85%
in 5 days with regular addition of ATP (1 eq in total) (data not
shown). The corresponding phosphorylated product was pu-
rified by semi-preparative HPLC in 72% yield. The same re-
action conditions, with the appropriate amount of enzyme
ScrKmitis, were applied to the synthesis of the phosphorylated
derivatives of aldoses D-altrose, D-glucose, D-mannose, D-
talose and ketoses D-psicose, L-sorbose, and D-tagatose. All
the phosphorylated products were formed in 30 min to 30 h
with at least 90% conversion except for D-psicose for which
4 days with 10 mM of ATP were required to reach 70% con-
version (Table 3). The corresponding phosphorylated prod-
ucts were purified by semi-preparative HPLC or ion-
exchange chromatography in 51 to 77% yield.
Biocatalytic synthesis and characterization
of phosphorylated hexoses
To evaluate the ScrKmitis ability for the synthesis of phosphor-
ylated hexoses, biocatalytic transformations of a representa-
tive panel of substrates were carried out on a 150-μmol scale.
In a first step, the biocatalytic synthesis was developed on D-
fructose. Polyphosphate (from 1 to 10 equivalents) was eval-
uated as low cost phosphate donor but no conversion could be
detected. Therefore, ATP was maintained using a recycling
system. Regeneration of 0.5 mM of ATP by the PEP/
pyruvate kinase system led to a total conversion of 20 mM
110,0
100,0
90,0
80,0
70,0
60,0
50,0
40,0
30,0
Fig. 3 Temperature activity
profile of ScrKmitis. Error bars
represent the standard deviation
of three independent experiments
0
20
40
60
80
100
temperature (°C)

Appl Microbiol Biotechnol
120,00
100,00
80,00
60,00
40,00
20,00
0,00
Fig. 4 Effect of the metal ions on
the specific activity of ScrKmitis.
Error bars represent the standard
deviation of three independent
experiments
MgCl2 MnCl2 CoCl2
NiCl2
ZnCl2
CuCl2
CaCl2 no metal
ion
The phosphorylated sugars synthetized were all fully char-
acterized by mass spectrometry and NMR spectroscopy (Fig.
5). The regioselectivity of the hexose phosphorylation cata-
lyzed by ScrKmitis, was determined by NMR. Considering the
literature, phosphorylation was expected in position 6 (Conejo
et al. 2010). Indeed, 1H and 13C NMR spectra (Fig. S5–S10)
confirmed a C6 regioselectivity of the phosphorylation
(Table 3, entries 1–2, 4–8) except for L-sorbose (Table 3,
entry 3). A position of phosphorylation elsewhere than in
position 6 was also reinforced by a fragmentation in FTMS
different from those obtained for the other synthesized phos-
phorylated hexoses (Fig. S17). The observed coupling for C4
and C5 on 13C NMR spectra suggested a phosphorylation at
sugar kinases. These enzymes shared the specific signatures of
the ROK family originally built from only few members, signa-
tures which were thereby reinforced by the large set of experi-
mentally tested kinases. Notably, we confirmed that the ATP/
Mg2+ binding site of fructokinases was EXGXT and not
DXGXT (Conejo et al. 2010) as for other ROK-type sugar
kinases. However, the biological function of Mak from E. coli
(Uniprot ID: P23917) described as fructokinase (Miller and
Raines 2004) could be questioned since it is not clusterized with
fructokinases in the phylogenetic tree and its ATP/Mg2+ binding
site is DXGXT. These features consolidated the signatures pre-
dicted to partition fructokinases from other sugar kinases.
The kinases from this study were active toward various
sugars and many of them were found thermotolerant. The
combined analysis of the phylogeny including a careful inven-
tory of ROK-type kinases, and our experimental data, re-
vealed that the majority of the 61 sugar kinases from this study
are mannofructokinases not related to the already known
ROK-type sugar kinases, mostly described as glucokinases
(21 out of the 27 known ROK-type sugar kinases according
to the inventory Table S4 and Table S5). As underlined by
Conejo and co-workers, carbohydrate specificity of individual
ROK-type kinase is difficult to assign due to the lack of ex-
perimental data (Conejo et al. 2010). In this study, we exper-
imentally assigned the substrate specificity of 20 ATP-
dependent fructokinases and 5 ATP-dependent glucokinases
previously annotated as ROK family proteins. In addition, we
experimentally confirmed the predicted annotations for 23
fructokinases and 3 glucokinases. Finally, function could be
assigned without ambiguity for 7 Buncharacterized proteins^
revealing 6 mannofrucokinases and 1 glucokinase.
1
the 5-position which was confirmed by a 2D H-31P NMR
analysis showing a coupling between 31P and 1H5 (Fig. S18).
Discussion
The exploration of the protein natural diversity is one of the most
efficient strategies to provide enzymes possessing new or im-
proved properties. In this study, we experimentally identified 61
Table 2 Kinetic parameters of ScrKmitis
Entry Substrate
k
cat (s−1
)
KM (μM)
k )
cat/KM(s−1 M−1
1
2
3
4
5
6
7
8
9
ATP
186
2
45.0 2.8
4.1 106
1.5 106
2.7 105
1.1 104
3.5 102
3.4 102
1.4 102
57
D-mannose 223 10 150.0 29.0
D-fructose 193 10 730.0 63.0
D-altrose
D-tagatose
D-talose
163
6
15.0 103 1.6 103
45 1.3 13.0 103 1.5 103
9.5 0.3 28.0 103 3.3 103
3.3 0.1 23.0 103 2.8 103
2.7 0.1 46.9 103 3.8 103
1.4 0.1 168.0 103 16.5 103
Little is known about the substrate scope of ROK-type
kinases. It was generally restricted to D-fructose, D-glucose,
and D-mannose. Only few were reported for other hexoses,
even if some ROK-type glucokinases with broad substrate
specificity were described (Table S5). To address this lack of
D-glucose
L-sorbose
D-sorbose
9

Appl Microbiol Biotechnol
Fig. 5 Phosphorylated hexoses biocatalytically synthetized in this study
experimental data, we selected one promiscuous kinase
(ScrKmitis) and undertook an in-depth kinetic parameters and
substrate spectrum study. For this purpose, we determined its
specific activities for all hexoses (aldoses and ketoses D- and
L- series) and also some C4 and C5 monosaccharides.
ScrKmitis was clearly distinguished from those described in
the literature by its high catalytic efficiency toward both D-
mannose and D-fructose and was found to be the first purified
mannofructokinase deeply characterized from the ROK fam-
ily (Table 4). FrKmobilis, the only one other purified enzyme
with characterized manno- and fructokinase activity, shares
38% of sequence identity with ScrKmitis but showed only
moderate activity toward D-mannose compared to ScrKmitis.
Indeed, even if their Michaelis constants KM for D-fructose
and D-mannose were similar, Vmax of ScrKmitis for D-
mannose was 500 times higher (Table 4). Furthermore, its
catalytic activity toward other hexoses as D-tagatose and un-
natural monosaccharides (D-altrose and D-talose) revealed its
large substrate scope mostly on hexoses. As far as we know,
such substrate promiscuity was never reported for any ROK-
type sugar kinase (Table S4 and Table S5).
In addition, as far as we know, ScrKmitis is the only purified
fructokinase encoded by an identified gene for which operat-
ing conditions have been so extensively studied (Table 4).
Table 3 Biocatalytic phosphorylation of some hexoses by ScrKmitis
Entry
Products
Conversion (%) (time)
Isolated yield (%)
Present forms (%)a
α-Furanose
β-Furanose
α-Pyranose
β-Pyranose
1
2
3
4
5
6
7
8
D-fructose-6P
D-psicose-6P
L-sorbose-5P
D-altrose-6P
D-tagatose-6P
D-glucose-6P
D-mannose-6P
D-talose-6P
> 99 (30 min)
70 (4 days)
> 99 (24 h)
90 (10 h)
72
51
67
57
51
56
77
77
21
35
nd
15
25
nd
nd
13
79
65
nd
11
75
nd
nd
10
nd
nd
nd
nd
43
nd
65
34
36
nd
> 95
31
> 99 (2 h)
> 99 (8 h)
> 99 (15 h)
95 (7 h)
nd
35
66
41
nd not detected
a As deduced from 13 C NMR spectra

Appl Microbiol Biotechnol
Table 4 Kinetic constants and optimal conditions of fructokinases from ROK family
D-fructose
D-mannose
Optimal
Gene
name
Enzyme
purity
Vmax
(U/mg
protein)
Km
(mM)
Vmax
(U/mg
protein)
Km
(mM)
T
(°C)
pH
Cations
References
This study
ScrKmitis
FrKmobilis
P
P
39
0.73
0.7
90
0.15
55
7–10
Mg2+ > Co2+ > Mn2+
>
Zn2+ > Ca2+ > Ni2+
0.35
(Scopes et al. 1985;
Zembrzuski
et al. 1992)
FrKmobilis
GmuE
P
0.21
0.75
0.38
0.77
nd
0.17
0.65
1
(Weisser et al. 1996)
(Nocek et al. 2011)
(Sato et al. 1993)
P
0.18
0.02 10−3
ScrKmutans
Mak
CE
CE
0.05 10−4
nd
(Miller and Raines
2004; Sproul
et al. 2001)
Q09124*
Q09123*
PH
PH
0.2
0.31
0.4
nd
nd
nd
7–8
Mg2+ > Co2+ > Fe2+ > Mn2+
Ni2+ > Zn2+ > Cd2+
>
(Thompson
et al. 1991)
0.07
~ 0.07
6.5–7.5
Mg2+ > Fe2+ > Co2+ > Mn2+
>
(Thompson
et al. 1992)
Ni2+ > Cd2+ > Zn2+ > Ca2+
One unit (U) of enzyme activity was defined as the amount of enzyme that catalyzed the conversion of 1 mM of substrate per minute
nd not determined but activity detected, P purified protein, CE crude extract, PH purified to homogeneity
*Entries are N terminal fragment proteins that are orphan of genes. For these fragments, a BLAST against Uniprot database showed 100% of local
identity with N terminal fragment of fructokinases from ROK family.
ScrKmitis kept more than 80% of activity between pH 7 and
10, and still 60% at pH 6–7 and pH 10–11. Such broader pH
range seems not shared by the two fructokinases orphan of
genes (Table 4). Its metal ion requirement is in accordance
with literature reported for ROK-type kinase, exemplified by
the clear visualization of the binding mode between the en-
zyme and divalent cation in crystal structures (Nishimasu et al.
2007; Nocek et al. 2011). Mg2+, Co2+, and Mn2+ were found
more effective on fructokinase activity of ScrKmitis than Zn2+,
Ca2+, and Ni2+. This tolerance with regard to a wide range of
bivalent metal ions was similar to those reported (Table 4). In
addition, despite coming from a mesophilic bacterium, it
showed a very broad temperature range (> 85% activity be-
tween 40 and 70 °C). Maximum activity around 40–50 °C was
reported for kinases from mesophilic bacteria, like hexokinase
from the Arthrobacter sp. (Mukai et al. 2003) (45 °C) and
glucokinase from Streptomyces peucetius var. caesius
(Ramos et al. 2004) (42 °C) but without data above 50 °C
reported (Table S5). ScrKmitis seemed to distinguish from
these reported enzymes by its conserved activities at 60–
70 °C and from kinases from thermophilic organisms, which
generally exhibit a restricted maximum activity temperature
range (70 °C to 100 °C) (Dorr et al. 2003; Hansen et al. 2002;
Qian et al. 2013). Interestingly, ScrKmitis thermotolerance
allowed its purification by a simple cell crude extract heat
treatment with however 20 to 30% loss of activity. By com-
parison, ScrK from Lactobacillus lactis is completely
inactivated by heating treatment within 3 min at 65 °C
(Thompson et al. 1991). It is noteworthy that from an indus-
trial point of view, enzymatic processes at elevated tempera-
ture offer many advantages, such as higher reaction rate, im-
proved solubility of organic substrates, and higher tolerance
toward nonconventional media (Choi et al. 2015; Elleuche
et al. 2014). In addition, thermostable enzymes offer better
resistance against protein destabilizing factors during protein
engineering processes (Littlechild 2015).
We demonstrated the biocatalytic applicability of this sugar
kinase from Streptococcus mitis by synthesizing 8 phosphor-
ylated hexoses through simple ATP-dependent phosphoryla-
tion and in situ regeneration of ATP by PEP/PK system. All
the substrates were regioselectively phosphorylated in posi-
tion 6 except L-sorbose which gave the 5-derivative. One hy-
pothesis could be that the reacting conformational form in
the active site is the pyranose one which eliminates any
possibility of phosphorylation in position 6, supported by
structures of gluco- and fructokinases showing closed con-
formational forms of the substrates in the active sites
(Nishimasu et al. 2007; Nocek et al. 2011). In addition,
Balieu and collaborators have observed the preferred pyra-
nose form in water for L-sorbose (Zebiri et al. 2011). We
observed a trend that the enzyme better converts aldoses with
S-configuration at position 2 (α- or β –pyranose configura-
tion) with preferentially R configuration at positions 4 (spe-
cific activity(D-altrose and D-mannose) > specific activity(D-talose)),

Appl Microbiol Biotechnol
substrate. Green Chem 19:519–526. https://doi.org/10.1039/
C6GC02652D
as well as ketoses with S configuration at position 3, with
preferentially R configuration at position 4 (specific
activity(D-fructose) > specific activity(D-tagatose)).
All these results revealed ScrKmitis as an Beasy enzyme^
(Bornscheuer 2016), efficient and versatile biocatalyst for the
synthesis of phosphorylated sugars, compounds of particular
interest in the study of various metabolic diseases
(Wohlgemuth et al. 2017).
Desvergnes S, Courtiol-Legourd S, Daher R, Dabrowski M, Salmon L,
Therisod M (2012) Synthesis and evaluation of malonate-based in-
hibitors of phosphosugar-metabolizing enzymes: class II fructose-1,
6-bis-phosphate aldolases, type I phosphomannose isomerase, and
phosphoglucose isomerase. Bioorg Med Chem 20(4):1511–1520.
https://doi.org/10.1016/j.bmc.2011.12.050
Dorr C, Zaparty M, Tjaden B, Brinkmann H, Siebers B (2003) The
hexokinase of the hyperthermophile Thermoproteus tenax. ATP-
dependent hexokinases and ADP-dependent glucokinases, two al-
ternatives for glucose phosphorylation in Archaea. J Biol Chem
278(21):18744–18753. https://doi.org/10.1074/jbc.M301914200
Elleuche S, Schröder C, Sahm K, Antranikian G (2014) Extremozymes—
biocatalysts with unique properties from extremophilic microorgan-
isms. Curr Opin Biotechnol 29:116–123. https://doi.org/10.1016/j.
copbio.2014.04.003
Acknowledgments The authors would like to thank Jean-François
1
Gallard (ICSN, France) for H-31P NMR experiments, Olek Maciejak
and Marie-Jeanne Clément (SABNP, INSERM U 1204—Université
d’Evry Val-d’Essonne, Université Paris-Saclay, France) for assistance in
1H and 13C NMR experiments, the Region Ile de France for financial
support of the 600 MHz NMR spectrometer, Alain Perret for fruitful
discussions and proofreading of the manuscript, and Christine Pelle and
Peggy Sirvain for nickel affinity chromatography purification of
ScrKmitis. We thank C. Rivoire (the Swiss-Prot Group at the SIB Swiss
Institute of Bioinformatics) for earlier Uniprot identifier assignments.
Franke FP, Kapuscinski M, Lyndon P (1985) Synthesis and n.m.r. char-
acterization of intermediates in the L-type pentose phosphate cycle.
Carbohydr Res 143:69–76. https://doi.org/10.1016/S0008-6215(00)
90696-7
Guérard-Hélaine C, Debacker M, Clapés P, Szekrenyi A, Hélaine V,
Lemaire M (2014) Efficient biocatalytic processes for highly valu-
able terminally phosphorylated C5 to C9 D-ketoses. Green Chem
16(3):1109–1113. https://doi.org/10.1039/c3gc42140f
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Guérard-Hélaine C, de Berardinis V, Besnard-Gonnet M, Darii E,
Debacker M, Debard A, Fernandes C, Hélaine V, Mariage A,
Pellouin V, Perret A, Petit J-L, Sancelme M, Lemaire M,
Salanoubat M (2015) Genome mining for innovative biocatalysts:
new dihydroxyacetone aldolases for the chemist’s toolbox.
ChemCatChem 7(12):1871–1879. https://doi.org/10.1002/cctc.
201500014
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Hansen T, Reichstein B, Schmid R, Schönheit P (2002) The first Archaeal
ATP-dependent glucokinase, from the hyperthermophilic
crenarchaeon Aeropyrum pernix, represents a monomeric, extremely
thermophilic ROK glucokinase with broad hexose specificity. J
Bacteriol 184(21):5955–5965. https://doi.org/10.1128/jb.184.21.
5955-5965.2002
Heinonen JK, Lahti RJ (1981) A new and convenient colorimetric deter-
mination of inorganic orthophosphate and its application to the assay
of inorganic pyrophosphatase. Anal Biochem 113(2):313–317.
https://doi.org/10.1016/0003-2697(81)90082-8
Hélaine V, Mahdi R, Sudhir Babu GV, de Berardinis V, Wohlgemuth R,
Lemaire M, Guérard-Hélaine C (2015) Straightforward synthesis of
terminally phosphorylated L-sugars via multienzymatic cascade re-
actions. Adv Synth Catal 357(8):1703–1708. https://doi.org/10.
1002/adsc.201500190
References
Bastard K, Smith AA, Vergne-Vaxelaire C, Perret A, Zaparucha A, De
Melo-Minardi R, Mariage A, Boutard M, Debard A, Lechaplais C,
Pelle C, Pellouin V, Perchat N, Petit JL, Kreimeyer A, Medigue C,
Weissenbach J, Artiguenave F, De Berardinis V, Vallenet D,
Salanoubat M (2014) Revealing the hidden functional diversity of
an enzyme family. Nat Chem Biol 10(1):42–49. https://doi.org/10.
1038/nchembio.1387
Bornscheuer UT (2016) Biocatalysis: successfully crossing boundaries.
Angew Chem Int Ed Eng 55(14):4372–4373. https://doi.org/10.
1002/anie.201510042
Charmantray F, Hélaine V, Legeret B, Hecquet L (2009) Preparative scale
enzymatic synthesis of D-sedoheptulose-7-phosphate from β-
hydroxypyruvate and D-ribose-5-phosphate. J Mol Catal B Enzym
57(1–4):6–9. https://doi.org/10.1016/j.molcatb.2008.06.005
Choi JM, Han SS, Kim HS (2015) Industrial applications of enzyme
biocatalysis: current status and future aspects. Biotechnol Adv 33:
1443–1454. https://doi.org/10.1016/j.biotechadv.2015.02.014
Conejo MS, Thompson SM, Miller BG (2010) Evolutionary bases of
carbohydrate recognition and substrate discrimination in the ROK
protein family. J Mol Evol 70(6):545–556. https://doi.org/10.1007/
s00239-010-9351-1
Davids T, Schmidt M, Böttcher D, Bornscheuer UT (2013) Strategies for
the discovery and engineering of enzymes for biocatalysis. Curr
Opin Chem Biol 17(2):215–220. https://doi.org/10.1016/j.cbpa.
2013.02.022
de Berardinis V, Guérard-Hélaine C, Darii E, Bastard K, Hélaine V,
Mariage A, Petit JL, Poupard N, Sánchez-Moreno I, Stam M,
Gefflaut T, Salanoubat S, Lemaire M (2017) Expanding the reaction
space of aldolases using hydroxypyruvate as a nucleophilic
Holmes KC, Sander C, Valencia A (1993) A new ATP-binding fold in
actin, hexokinase and Hsc70. Trends Cell Biol 3(2):53–59. https://
doi.org/10.1016/0962-8924(93)90161-S
Howe K, Bateman A, Durbin R (2002) QuickTree: building huge
neighbour-joining trees of protein sequences. Bioinformatics
18(11):1546–1547. https://doi.org/10.1093/bioinformatics/18.11.
1546
Jaworek D, Welsch J (1985) Adenosine 5-diphosphate and adenosine 5-
monophosphate. In: Bergmeyer HU, Bergmeyer J, Graßl M (eds)
Methods Enz Anal, vol 7, pp 365–370
Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel method
for rapid multiple sequence alignment based on fast Fourier trans-
form. Nucleic Acids Res 30(14):3059–3066
Koerner Jr TA, Voll RJ, Cary LW, Younathan ES (1980) Carbon-13
nuclear magnetic resonance studies and anomeric composition of
ketohexose phosphates in solution. Biochemistry 19(12):2795–
2801. https://doi.org/10.1021/bi00553a040
Kreimeyer A, Perret A, Lechaplais C, Vallenet D, Medigue C, Salanoubat
M, Weissenbach J (2007) Identification of the last unknown genes in

Appl Microbiol Biotechnol
the fermentation pathway of lysine. J Biol Chem 282(10):7191–
7197. https://doi.org/10.1074/jbc.M609829200
Kreuzer P, Gartner D, Allmansberger R, Hillen W (1989) Identification
and sequence analysis of the Bacillus subtilis W23 xylR gene and
xyl operator. J Bacteriol 7(171):3840–3845. https://doi.org/10.1021/
bi00553a040
Larion M, Moore LB, Thompson SM, Miller BG (2007) Divergent evo-
lution of function in the ROK sugar kinase superfamily: role of
enzyme loops in substrate specificity. Biochemistry 46(47):13564–
13572. https://doi.org/10.1021/bi700924d
Sato Y, Yamamoto Y, Kizaki H, Kuramitsu HK (1993) Isolation, charac-
terization and sequence analysis of the scrK gene encoding
fructokinase of Streptococcus mutans. J Gen Microbiol 139(5):
921–927. https://doi.org/10.1099/00221287-139-5-921
Schorken U (1998) Thiamin-dependent enzymes as catalysts in
chemoenzymatic syntheses. Biochim Biophys Acta 1385(E 2):229–243
Scopes RK, Testolin V, Stoter A, Griffiths-Smith K, Algar EM (1985)
Simultaneous purification and characterization of glucokinase,
fructokinase and glucose-6-phosphate dehydrogenase from
Zymomonas mobilis. Biochem J 228(3):627–634
Letunic I, Bork P (2016) Interactive tree of life (iTOL) v3: an online tool for
the display and annotation of phylogenetic and other trees. Nucleic
Acids Res 44(W1):W242–W245. https://doi.org/10.1093/nar/gkw290
Littlechild JA (2015) Enzymes from extreme environments and their
industrial applications. Front Bioeng Biotechnol 3:161. https://doi.
org/10.3389/fbioe.2015.00161
Miller BG, Raines RT (2004) Identifying latent enzyme activities: substrate
ambiguity within modern bacterial sugar kinases. Biochemistry 43(21):
6387–6392. https://doi.org/10.1021/bi049424m
Mukai T, Kawai S, Matsukawa H, Matuo Y, Murata K (2003)
Characterization and molecular cloning of a novel enzyme, inorgan-
ic polyphosphate/ATP-glucomannokinase, of Arthrobacter sp. strain
KM. Appl Environ Microbiol 69(7):3849–3857. https://doi.org/10.
1128/aem.69.7.3849-3857.2003
Nishimasu H, Fushinobu S, Shoun H, Wakagi T (2007) Crystal structures of
an ATP-dependent hexokinase with broad substrate specificity from the
hyperthermophilic archaeon Sulfolobus tokodaii. J Biol Chem 282(13):
9923–9931. https://doi.org/10.1074/jbc.M610678200
Nocek B, Stein AJ, Jedrzejczak R, Cuff ME, Li H, Volkart L, Joachimiak A
(2011) Structural studies of ROK fructokinase YdhR from Bacillus
subtilis: insights into substrate binding and fructose specificity. J Mol
Biol 406(2):325–342. https://doi.org/10.1016/j.jmb.2010.12.021
Plumbridge J (2001) DNA binding sites for the Mlc and NagC proteins:
regulation of nagE, encoding the N-acetylglucosamine-specific
transporter in Escherichia coli. Nucleic Acids Res 29(2):506–514.
https://doi.org/10.1093/nar/29.2.506
Postma PW, Lengeler JW, Jacobson GR (1993) Phosphoenolpyruvate:
carbohydrate phosphotransferase systems of bacteria. Microbiol
Rev 3(57):543–594
Qian Z, Zhao J, Bai X, Tong W, Chen Z, Wei H, Wang Q, Liu S (2013)
Thermal stability of glucokinases in Thermoanaerobacter
tengcongensis. Biomed Res Int 2013:646539. https://doi.org/10.
1155/2013/646539
Shaeri J, Wright I, Rathbone EB, Wohlgemuth R, Woodley JM (2008)
Characterization of enzymatic D-xylulose 5-phosphate synthesis.
Biotechnol Bioeng 101(4):761–767. https://doi.org/10.1002/bit.21949
Sizemore C, Buchner E, Rygus T, Witke C, Gotz F, Hillen W (1991)
Organization, promoter analysis and transcriptional regulation of
the Staphylococcus xylosus xylose utilization operon. Mol Gen
Genet 227(3):377–384. https://doi.org/10.1007/BF00273926
Solovjeva ON, Kochetov GA (2008) Enzymatic synthesis of d-xylulose
5-phosphate from hydroxypyruvate and d-glyceraldehyde-3-phos-
phate. J Mol Catal B Enzym 54(3–4):90–92. https://doi.org/10.
1016/j.molcatb.2007.12.016
Sproul AA, Lambourne LT, Jean-Jacques DJ, Kornberg HL (2001) Genetic
control of manno(fructo)kinase activity in Escherichia coli. PNAS
98(26):15257–15259. https://doi.org/10.1073/pnas.211569798
Thompson J, Sackett DL, Donkersloot JA (1991) Purification and properties
of fructokinase I from Lactococcus lactis. Localization of scrK on the
sucrose-nisin transposon Tn5306. J Biol Chem 33(266):22626–22633
Thompson J, Nguyen NY, Robrish SA (1992) Sucrose fermentation by
Fusobacterium mortiferum ATCC 25557: transport, catabolism, and
products. J Bacteriol 10(174):3227–3235
Titgemeyer F, Reizer J, Reizer A, Saier MH Jr (1994) Evolutionary rela-
tionships between sugar kinases and transcriptional repressors in
bacteria. Microbiology 140(Pt 9):2349–2354. https://doi.org/10.
1099/13500872-140-9-2349
Uehara T, Park JT (2004) The N-acetyl-D-glucosamine kinase of
Escherichia coli and its role in murein recycling. J Bacteriol 186(21):
7273–7279. https://doi.org/10.1128/JB.186.21.7273-7279.2004
Vergne-Vaxelaire C, Bordier F, Fossey A, Besnard-Gonnet M, Debard A,
Mariage A, Pellouin V, Perret A, Petit J-L, Stam M, Salanoubat M,
Weissenbach J, De Berardinis V, Zaparucha A (2013) Nitrilase ac-
tivity screening on structurally diverse substrates: providing biocat-
alytic tools for organic synthesis. Adv Synth Catal 355(9):1763–
1779. https://doi.org/10.1002/adsc.201201098
Ramos I, Guzman S, Escalante L, Imriskova I, Rodriguez-Sanoja R,
Sanchez S, Langley E (2004) Glucose kinase alone cannot be re-
sponsible for carbon source regulation in Streptomyces peucetius
var. caesius. Res Microbiol 155(4):267–274. https://doi.org/10.
1016/j.resmic.2004.01.004
Weisser P, Kramer R, Sprenger GA (1996) Expression of the Escherichia
coli pmi gene, encoding phosphomannose-isomerase in Zymomonas
mobilis, leads to utilization of mannose as a novel growth substrate,
which can be used as a selective marker. Appl Environ Microbiol
11(62):4155–4161
Rother C, Gutmann A, Gudiminchi R, Weber H, Lepak A, Nidetzky B
(2017) Biochemical characterization and mechanistic analysis of the
levoglucosan kinase from Lipomyces starkeyi. Chembiochem 19:
596–603. https://doi.org/10.1002/cbic.201700587
Rudolph FB, Fromm HJ (1971) A study on the kinetics and mechanism of D-
lyxose and D-xylose activation of the adenosine triphosphatase activity
associated with yeast hexokinase. J Biol Chem 7(246):2104–2110
Sánchez-Moreno I, Hélaine V, Poupard N, Charmantray F, Légeret B,
Hecquet L, García-Junceda E, Wohlgemuth R, Guérard-Hélaine C,
Lemaire M (2012) One-pot cascade reactions using fructose-6-
phosphate aldolase: efficient synthesis of D-arabinose 5-phosphate,
D-fructose 6-phosphate and analogues. Adv Synth Catal 354(9):
1725–1730. https://doi.org/10.1002/adsc.201200150
Wohlgemuth R (2009) C2-Ketol elongation by transketolase-catalyzed
asymmetric synthesis. J Mol Catal B Enzym 61(1–2):23–29.
https://doi.org/10.1016/j.molcatb.2009.04.001
Wohlgemuth R, Liese A, Streit W (2017) Biocatalytic phosphorylations
of metabolites: past, present, and future. Trends Biotechnol 35(5):
452–465. https://doi.org/10.1016/j.tibtech.2017.01.005
Zebiri I, Balieu S, Guilleret A, Reynaud R, Haudrechy A (2011) The
chemistry of L-Sorbose. Eur J Org Chem 2011(16):2905–2910.
https://doi.org/10.1002/ejoc.201001578
Zembrzuski B, Chilco P, Liu XL, Liu J, Conway T, Scopes R (1992)
Cloning, sequencing, and expression of the Zymomonas mobilis
fructokinase gene and structural comparison of the enzyme with
other hexose kinases. J Bacteriol 11(174):3455–3460
Sato Y, Poy F, Jacobson GR, Kuramitsu HK (1989) Characterization and
sequence analysis of the scrA gene encoding enzyme IIScr of the
Streptococcus mutans phosphoenolpyruvate-dependent sucrose
phosphotransferase system. J Bacteriol 1(171):263–271. https://
doi.org/10.1128/JB.171.1.263-271
Zhao S, Kumar R, Sakai A, Vetting MW, Wood BM, Brown S, Bonanno
JB, Hillerich BS, Seidel RD, Babbitt PC, Almo SC, Sweedler JV,
Gerlt JA, Cronan JE, Jacobson MP (2013) Discovery of new en-
zymes and metabolic pathways using structure and genome context.
Nature 502(7473):698–702. https://doi.org/10.1038/nature12576