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

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-Hélaine^2,3& Ekaterina Darii¹& Adrien Debard¹& Stessy Nepert¹& Virginie Pellouin¹

&

Marielle Lemaire^2,3& 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 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 10⁶s⁻¹M⁻¹and 2.7 10⁵s⁻¹M⁻¹, respectively, but also significant ones toward D-tagatose (3.5 10²s⁻¹M⁻¹) and the

unnatural monosaccharides ^D-altrose (1.110⁴s⁻¹M⁻¹) and D-talose (3.4 10²s⁻¹M⁻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 ScrK_mitiswere

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 (ScrK_mitis

)

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 ScrK_mitiswas 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 A₃₄₀using 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⁻¹cm⁻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 ScrK_mitis

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 ScrK_mitisat 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

ScrK_mitisdetermined 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 ScrK_mitis

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 ScrK_mitis.

Kinetic constants were determined on purified

ScrK_mitisby 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 ScrK_mitis. 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 ScrK_mitis. 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 ScrK_mitistoward various substrates.

Specific activities of ScrK_mitiswere determined by using

protocol A with MgCl₂as 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 ScrK_mitis. Thermotolerance of

ScrK_mitiswas 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 ScrK_mitis

was measured with protocol A for D-fructose, D-man-

nose, and ^D-glucose.

mercial ATP, followed by addition of ScrK_mitis

.

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 MgCl_2,12 μg of purified

ScrK_mitis, 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

ScrK_mitis. 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 ScrK_mitisversus 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 ScrK_mitisversus 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 ScrK_mitisversus 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 T_eva= 70 °C; T_neb= 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)₂CO₃10 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 NH₄HCO₃buffer 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 NH₄HCO₃concentrations: 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 NH₄HCO₂20 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 ScrK_mitisin 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 ScrK_mitisin 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 ScrK_mitisin 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 ScrK_mitis

.

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

ScrK_mitis. 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 ScrK_mitisin 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 ScrK_mitisin 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 ScrK_mitisin 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 ScrK_mitisin 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 ScrK_mitisin 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 (ScrK_mutants, Uniprot ID:

Q07211) involved in the sucrose transport through a

phosphoenolpyruvate-dependent phosphotransferase system

(PTS) (Sato et al. 1993). An apparent V_maxfor 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 ScrK_mutantsshare 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

ScrK_mitis. Purified ScrK_mitiswas 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 ScrK_mitisis a homodimer, as

previously reported for the fructokinase of Zymomonas

mobilis (FrK_mobilis) (Scopes et al. 1985). Biochemical proper-

ties of this purified enzyme were as follows.

(i) Substrate specificity

To determine the substrate range of ScrK_mitis, 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. ScrK_mitisshowed 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⁻¹of protein) were

measured for all natural hexoses except ^D-galactose. ScrK_mitis

phosphorylated also rare sugars, like for D-psicose, D-allose,

and ^D-gulose, with low specific activities (< 0.1 U mg⁻¹of

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 ScrK_mitistoward hexoses (D- and

L-series)

corresponding to the expected mass of ScrK_mitis(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 ScrK_mitispurified 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

< 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. ScrK_mitisdisplayed 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

ScrK_mitisfrom 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, ScrK_mitisshowed a low ATPase activity in our

conditions (K_M= 0.9 mM; k_cat/K_M= 32 s⁻¹M⁻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

ScrK_mitismetal 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⁻¹of enzyme (Table 1).

Lowest K_Mwere 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). ScrK_mitiswas highly effec-

tive toward ^D-mannose and D-fructose (k_cat/K_M= 1.5 10⁶and

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 ScrK_mitisat 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 ScrK_mitis

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

ScrK_mitis, 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 ScrK_mitisability 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 ScrK_mitis.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 ScrK_mitis.

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 ScrK_mitis, 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 ScrK_mitis

Entry Substrate

k

_cat(s⁻¹

)

K_M(μM)

k )

_cat/K_M(s⁻¹M⁻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 10³1.6 10³

45 1.3 13.0 10³1.5 10³

9.5 0.3 28.0 10³3.3 10³

3.3 0.1 23.0 10³2.8 10³

2.7 0.1 46.9 10³3.8 10³

1.4 0.1 168.0 10³16.5 10³

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

(ScrK_mitis) 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.

ScrK_mitiswas 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). FrK_mobilis, the only one other purified enzyme

with characterized manno- and fructokinase activity, shares

38% of sequence identity with ScrK_mitisbut showed only

moderate activity toward ^D-mannose compared to ScrK_mitis.

Indeed, even if their Michaelis constants K_Mfor D-fructose

and ^D-mannose were similar, Vmax of ScrK_mitisfor 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, ScrK_mitisis 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 ScrK_mitis

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

21

35

nd

15

25

nd

13

79

65

nd

11

75

nd

10

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

^aAs deduced from ¹³C 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

ScrK_mitis

FrK_mobilis

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)

FrK_mobilis

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

ScrK_mutans

Mak

CE

0.05 10⁻⁴

nd

(Miller and Raines

2004; Sproul

et al. 2001)

Q09124*

Q09123*

PH

0.2

0.31

0.4

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.

ScrK_mitiskept 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 ScrK_mitisthan 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). ScrK_mitisseemed 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, ScrK_mitisthermotolerance

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 ScrK_mitisas 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).

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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

ScrK_mitis. We thank C. Rivoire (the Swiss-Prot Group at the SIB Swiss

Institute of Bioinformatics) for earlier Uniprot identifier assignments.

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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,

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201500014

Ethical approval This article does not contain any studies with human

participants or animals performed by any of the authors.

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Article Doi

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

DOI: 10.1007/s00253-018-9018-1

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

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

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