Medium-chain dehydrogenases with new specificity: Amino mannitol dehydrogenases on the azasugar biosynthetic pathway

Article abstract of DOI:10.2174/092986652101131219093413

Azasugar biosynthesis involves a key dehydrogenase that oxidizes 2-amino-2-deoxy-D-mannitol to the 6-oxo compound. The genes encoding homologous NAD-dependent dehydrogenases from Bacillus amyloliquefaciens FZB42, B. atrophaeus 1942, and Paenibacillus polymyxa SC2 were codon-optimized and expressed in BL21(DE3) Escherichia coli. Relative to the two Bacillus enzymes, the enzyme from P. polymyxa proved to have superior catalytic properties with a V_max of 0.095 ± 0.002 μmol/min/mg, 59-fold higher than the B. amyloliquefaciens enzyme. The preferred substrate is 2-amino-2-deoxy-D- mannitol, though mannitol is accepted as a poor substrate at 3% of the relative rate. Simple amino alcohols were also accepted as substrates at lower rates. Sequence alignment suggested D283 was involved in the enzyme's specificity for aminopolyols. Point mutant D283N lost its amino specificity, accepting mannitol at 45% the rate observed for 2-amino-2-deoxy-D-mannitol. These results provide the first characterization of this class of zinc-dependent medium chain dehydrogenases that utilize aminopolyol substrates.

Full text of DOI:10.2174/092986652101131219093413

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10
Protein & Peptide Letters, 2014, 21, 10-14
Medium-Chain Dehydrogenases with New Specificity: Amino Mannitol
Dehydrogenases on the Azasugar Biosynthetic Pathway
Yanbin Wu, Jeffrey Arciola, and Nicole Horenstein*
Department of Chemistry, University of Florida, Gainesville Florida, 32611-7200, USA
Abstract: Azasugar biosynthesis involves a key dehydrogenase that oxidizes 2-amino-2-deoxy-D-mannitol to the 6-oxo
compound. The genes encoding homologous NAD-dependent dehydrogenases from Bacillus amyloliquefaciens FZB42, B.
atrophaeus 1942, and Paenibacillus polymyxa SC2 were codon-optimized and expressed in BL21(DE3) Escherichia coli.
Relative to the two Bacillus enzymes, the enzyme from P. polymyxa proved to have superior catalytic properties with a
Vmax of 0.095 0.002 ꢀmol/min/mg, 59-fold higher than the B. amyloliquefaciens enzyme. The preferred substrate is 2-
amino-2-deoxy-D-mannitol, though mannitol is accepted as a poor substrate at 3% of the relative rate. Simple amino alco-
hols were also accepted as substrates at lower rates. Sequence alignment suggested D283 was involved in the enzyme’s
specificity for aminopolyols. Point mutant D283N lost its amino specificity, accepting mannitol at 45% the rate observed
for 2-amino-2-deoxy-D-mannitol. These results provide the first characterization of this class of zinc-dependent medium
chain dehydrogenases that utilize aminopolyol substrates.
Keywords: Aminopolyol, azasugar, biosynthesis, dehydrogenase, mannojirimycin, nojirimycin.ꢀ
INTRODUCTION
are sufficient to convert fructose-6-phosphate into manno-
jirimycin [9]. We proposed that the gutB1 gene product was
responsible for the turnover of 2-amino-2-deoxy-D-mannitol
(2AM) into mannojirimycin as shown in Fig. 2. Feeding the
B. amyloliquefaciens gabT1 knockout with 2AM restored
azasugar production [9].
Azasugars such as the nojirimycins [1] are natural prod-
ucts that are analogs of monosaccharides that feature a nitro-
gen in the ring rather than oxygen (Fig. 1). In Nature they are
produced by various Bacillus, Streptomyces and plant spe-
cies [2-4]. Azasugars have served as the inspiration for the
synthesis of many different glycosidase inhibitors [5] and
have in recent times also enjoyed applications as chemical
chaperones for assisting in the folding and stabilization of
mutant enzymes responsible for lysosomal storage diseases
[6]. Examples include Miglustat, N-butyl-1-deoxynojiri-
mycin, for type I Gaucher’s disease [7] and Miglitol, (N-2-
hydroxyethyl)-1-deoxynojirimycin for modulation of post-
prandial bloodsugar in treatment of type II diabetes [8].
Sequence analyses indicate that GutB1 is a zinc-
dependent NAD(P)-dependent alcohol dehydrogenase, simi-
lar to sorbitol dehydrogenase, and is a member of the me-
dium-chain dehydrogenase/reductase (MDR) superfamily
[11]. As it was known that other species with sequenced ge-
nomes were deoxynojirimycin producers, we sought to iden-
tify and then compare other homologues possessing the
unique dehydrogenase activity demonstrated by the B. amy-
loliquefaciens enzyme. A protein BLAST analysis of the
three-gene azasugar biosynthetic signature found in B. amy-
loliquefaciens was conducted and identified similar ORFs in
Bacillus and related species. When we included more distant
hits we identified that Paenibacillus polymyxa SC2 has five
ORFs that appear to include the three functions we identified
in B. amyloliquefaciens, as well as an additional two
(PPSC2_c2587 and PPSC2_c2588) that code for putative
mannitol dehydrogenase and mannonate dehydratase activi-
ties that may also be part of the pathway [12]. In the work
we describe here, we characterize the GutB1 dehydrogenase
from B. amyloliquefaciens FZB42, the closely related en-
zyme from B. atrophaeus 1942 [13] (86% identity) and the
more distantly related homolog from Paenibacillus polymyxa
SC2 (33% identity). We show that the so-called GutB1 en-
zymes prefer substrates bearing an amino group, distinguish-
ing themselves from previously characterized polyol dehy-
drogenases.
Although azasugars have been known for quite some
time, the enzymatic machinery responsible for their synthesis
has only recently become the topic of experimental inquiry.
Further, the identity of enzymes in the entire pathway has not
yet been determined nor have any been characterized indi-
vidually. In this Letter we wish to report the expression and
functional characterization of a key dehydrogenase found in
azasugar biosynthetic clusters. Recent investigation of 1-
deoxynojirimycin biosynthesis has led to the identification of
Bacillus amyloliquefaciens genes gabT1, yktC1 and gutB1 as
part of the overall azasugar biosynthetic pathway [9,10].
These three genes respectively encode for transaminase,
phosphatase and dehydrogenase activity that we have shown
*Address correspondence to this author at the Department of Chemistry,
University of Florida, Gainesville Florida, 32611-7200, USA; Tel: 01-(352)-
392-9859; E-mail: horen@chem.ufl.edu
Wu, Y. Horenstein, N. Unpublished observations for recombinant YktC1
from B. amyloliquefaciens.
1875-5305/14 $58.00+.00
© 2014 Bentham Science Publishers

Medium-Chain Dehydrogenases with New Specificity
Protein & Peptide Letters, 2014, Vol. 21, No. 1 11
ꢁ
Figure 1. Structures of nojirimycin (NJ), mannojirimycin (MJ) and deoxynojirimycin (DNJ). Both NJ and DNJ have the gluco configuration
and MJ has the manno configuration.
HO HO HO
OH
NH
HO HO HO
HO
GutB1
O
HO
OH
HO
HO
NH2
NAD+ NADH
HO
NH2
HO
MJ
Figure 2. Conversion of 2-amino-2-deoxy-D-mannitol to mannojirimycin via GutB1 catalyzed oxidation. After oxidation, the acyclic ami-
noaldehyde is able to spontaneously cyclize to mannojirimycin (MJ).ꢀ
linker sequence as described above for the B. amyloliquefa-
ciens gutB1 construct.
MATERIALS AND METHODS
General. The gutB1 genes were synthesized and codon
optimized by GenScript. Escherichia coli DH5alpha and E.
coli BL21 (DE3) strains were obtained from Invitrogen and
Novagen respectively. The pET30a expression vector was
obtained from Novagen. Restriction endonucleases, T4 DNA
ligase and thermostable polymerases were purchased from
New England Biolabs. Isopropyl-ꢀ-D-thiogalactopyranoside
(IPTG), kanamycin and protein MW standards were pur-
chased from Fisher Scientific. Oligonucleotides were pur-
chased from Integrated DNA Technologies. DNA sequenc-
ing was performed at the University of Florida Sanger Se-
quencing core facility.
P. polymyxa D283N mutation. This point mutation was
made using the Q5 mutagenesis kit from New England Bio-
labs. The forward mutagenic primer sequence was 5’-
GAAGATTATCGGCTCAATTAACTCGCTGGGTACC-
TTCTC-3’. The reverse primer sequence was 5’-AGGCTG
CGATCAACCACTTCTTTCGGATTA-3’. The PCR ther-
mocycle was: 98 °C, 30 s; 25x (98 °C, 10 s; 66 °C, 30 s; 72
°C, 198 s), 72 °C, 120 s; followed by 4 °C, indefinite.
Protein expression and purification. The pET30a-
gutB1constructs were transformed into BL21 (DE3) E. coli.
Single colonies were selected to inoculate 5 mL overnight
starter cultures (Luria-Bertani medium containing 50 ꢀg/mL
kanamycin). Each starter culture was used to inoculate 500
ml of LB/K medium and the resulting cultures were grown
for 2-4 hours at 37 °C with constant shaking at 225 rpm.
Expression of GutB1 was induced by the addition of 0.5 mM
IPTG when an OD600 of 0.4 to 0.8 was reached. The cultures
were grown at 18 °C for 18 h with constant shaking at 225
rpm. The cells were harvested by centrifugation at 4 °C in a
GS3 rotor at 5000 rpm for 20 minutes. The cell pellets were
each resuspended in 20 mL of lysis buffer (20 mM Tris-HCl,
pH=7.9, 250 mM NaCl, 5 mM imidazole). After lysis by
French press and sonication, the lysate was centrifuged at
14,000 rpm for 30 min at 4 °C in an SS34 rotor. The clarified
lysate was applied to a Ni-IMAC resin (Qiagen), and the
column washed with a step gradient of 10, 50, and 100 mM
imidazole. GutB1 was eluted with 250 mM imidazole-
containing buffer (20 mM Tris-HCl, pH=7.9, 250 mM NaCl,
250 mM imidazole). The eluted enzyme was dialyzed at 4°C
against 20 mM Tris buffer (50 mM NaCl, pH=7.0).
Subcloning of the B. amyloliquefaciens gutB1 gene
into pET30a. The codon-optimized version of the gutB1
gene was originally prepared in pETBlue2. The pETBlue2-
gutB1 vector served as a template for PCR with the forward
primer 5’-GAGCCATGGGGATGAAAGCTCTGGTGTG
GAC-3’ and reverse primer 5’-AGAGCTCGAGTTACAGC
AGTTTCGGGTCGCTAAC-3’. The underlined sequences
correspond to the NcoI and XhoI restriction sites, in the for-
ward and reverse primers, respectively. The PCR product
was cloned into pET30a to generate the reconstruct plasmid
pET30a-opt-gutB1, and was verified by sequencing. This
construct codes for an N-terminal Met(His)6 tag fused to a
linker peptide with the following sequence upstream of the
native start Met: SSGLVPRGSGMKETAAAKFERQHMDS
PDLGTDDDDK.
Expression constructs for P. polymyxa SC2 and B.
atrophaeus gutB1 genes. The P. polymyxa (locus tag PPSC2
_c2584) and B. atrophaeus (locus tag BATR1942_19425)
gutB1 homologues were codon-optimized (Genscript) in
pUC57 with NcoI and XhoI sites flanking the start and stop
codons. The gutB1 genes were subcloned into the NcoI and
XhoI sites of the pET30 vector for expression. The recombi-
nant construct was confirmed by sequencing. These con-
structs afforded coded for the same N-terminal His tag and
Protein analyses. Protein was assayed by the Bradford
method using bovine serum albumin as a standard [14].
SDS-PAGE was performed at room temperature using 12%
acrylamide resolving gel and 5% acrylamide stacking gel.
Metal content was determined by inductively coupled

12 Protein & Peptide Letters, 2014, Vol. 21, No. 1
Wu et al.
plasma-mass spectrometry (ICP-MS) at the Chemical Analy-
sis Laboratory, University of Georgia, Athens. Samples were
exchanged into Chelex-treated 20 mM Tris HCl pH 7.0
buffer. Approximately 2.5 – 3 mg of protein in metal-free
buffer was further incubated with Chelex for 2 h at 4 °C to
remove weakly bound metals. A parallel blank without pro-
tein was prepared in the same way. The protein concentra-
tion was determined by careful Bradford assay and the solu-
tion was brought to a final volume in 1% HNO3 for analyses.
RESULTS AND DISCUSSION
The three GutB1 dehydrogenase genes from B. amyloliq-
uefaciens FZB42 (Bam), B. atrophaeus 1942 (Bat), and P.
polymyxa SC2 (Ppo) were obtained by synthesis in codon
optimized form. They readily expressed in E. coli as N-
terminal His-tagged fusion proteins in respective purified
yields of 80, 36, and 60 mg per liter of culture after Ni-
column chromatography. Electrophoretic analyses indicated
that we obtained near-homogeneous preparations of enzymes
at the anticipated approximate molecular weights. (Figures
S1-S3, supplemental information.) A MALDI-TOF analysis
of the Ppo enzyme yielded 42836 for [M+H] versus 42832
(predicted). Metal analysis of the Bam enzyme by ICP-MS
revealed a zinc/monomer stoichiometry of 2:1, consistent
with other members of the zinc-dependent MDR superfamily
often bearing two zincs per subunit. (Table S1, supplemental
information).
Synthesis of 2-amino-2-deoxy-D-mannitol (2AM) This
substrate was synthesized according to Liu et al [15], with
some modifications. The reaction involved reduction of D-
mannosamine (300 mg) with NaBH4 (572 mg in 5mL of
0.05M NaOH) for 1 hour at room temperature, at which time
TLC (silica, methanol:chloroform:water = 7:2:1, v/v/v) indi-
cated completion. Acetic acid was carefully added to destroy
the excess sodium borohydride. Methanol was added to the
quenched reaction mixture followed by rotary evaporation.
This was repeated four more times. The residue was taken up
in water (2 mL), the pH was adjusted to 5-6 and the solution
was applied to a Dowex-50 (H+) column (0.9 x 7 cm). The
resin was washed with 0.5 M HCl to elute Na+ (flame test),
then 2M HCl to elute the product 2AM. The product was
detected by a positive test with ninhydrin. Concentration in
We estimated steady-state kinetic parameters for the Bam
dehydrogenase by holding one substrate at high levels while
varying the other.(Figure S6 Supplemental information) The
Km for 2AM was estimated to be 5.5 1.6 mM, the Km for
NAD+ was 105 37 ꢀM with a kcat of 0.07 min-1. This very
slow turnover is consistent with results for GabT1 and
YktC11 enzymes from Bacillus amyloliquefaciens FZB42
which are also quite slow [17]. We determined that Bam
GutB1 can accept either NAD+ or NADP+, but does prefer
NAD+ since velocities measured with 2 mM NADP were
only 15 % of the rate observed with 2 mM NAD+(data not
shown). Initial examination of the Bat enzyme revealed it
had extremely low turnover like the Bam enzyme, a result
consistent with their sequence similarity. The Bat enzyme
was not characterized further. Interestingly the Ppo enzyme
exhibits significant sequence divergence from the Bam and
Bat homologs and we anticipated we might find different
kinetics for it. Initial assays for the enzyme indicated it was
considerably more active, and this was borne out in the ki-
netic analyses (Figure S7, supplemental information). The
Km for 2AM was estimated to be 1.7 0.1 mM, the Km for
NAD+ was 1.1 0.1 mM and kcat was 4.1 min-1. This turn-
over is approximately 59-fold higher than the Bam enzyme,
rendering it of interest for its possible ability to accept alter-
nate substrates. To this end various polyol and amino-alcohol
substrates were screened against the Bam and Ppo enzymes
for their activity relative to 2AM as presented in Table 1.
1
vacuo yielded 228 mg of 2AM (76 % yield). H-NMR and
13C-NMR indicated complete reduction to the desired 2AM.
(Supplemental information figures S4 and S5).
Enzymatic Assays. GutB1 enzymatic activity was moni-
tored by following NAD+ reduction at 340 nm. Assay reac-
tions were carried out at 25 °C, in a 1.00 mL standard reac-
tion mixture that contained 500 ꢀL 2X buffer (200 mM Tris,
50 mM NaCl, 5 ꢀM ZnCl2, pH = 8.57), and 50 ꢀlLBSA (20
mg/ml) for reactions involving the Bam enzyme. For estima-
tion of steady state kinetic parameters, one substrate was
held well above Km while the other substrate was varied. For
the Bam enzyme, the Km for NAD+ was estimated by holding
2AM at 16 mM while NAD+ was varied between 0.05 – 4
mM. The Km for 2AM was estimated by fixing the concen-
tration of NAD+ at 4 mM while varying 2AM between 0.16
– 53 mM. For the Ppo enzyme, 2AM was held at 53.3 mM
while NAD+ was varied between 0.01 – 10 mM. The concen-
tration of NAD+ was fixed at 4 mM while varying 2AM be-
tween 0.053 – 10.7 mM. Significant substrate inhibition was
observed when 2AM was assayed at 53 mM. All reactions
were initiated by the addition of 400 ꢀg of enzyme and
measured in triplicate. Data were acquired over 150 seconds
(Ppo) or 300 seconds (Bam). For substrate specificity stud-
ies, reactions were conducted in the same buffer system as
above. Test substrates were used at 10 mM concentration,
the NAD+ concentration was held at 2 mM, and reactions
were initiated with 180 ꢀg of enzyme. Analysis of the
D283N mutant kinetics used the standard reaction mixture
described above with 1.0 mg/mL BSA, 2AM (10 mM) or
mannitol (160 mM), NAD+ at 2 mM. Reactions were initi-
ated by addition of 100 ꢀg of either WT or D283N enzyme.
The data suggest several noteworthy characteristics of
this class of enzyme. Both enzymes accepted mannitol albeit
at reduced rates. Given that both rejected sorbitol, this indi-
cates that the manno configuration at C2 of the hexitol chain
is preferred. Interestingly, mannose, which exists primarily
as the ꢀ- pyranose in solution, was not accepted as a sub-
strate by either enzyme. This result suggests that acyclic sub-
strates are preferred, possibly indicating an extended binding
conformation. Fructose was not a substrate for the Bam en-
zyme; and it was not tested versus the Ppo dehydrogenase.
Interestingly the simple amino alcohols 4-amino-1-butanol
and 6-amino-1-hexanol were found to be alternate substrates
for the Ppo enzyme, but not the Bam enzyme. The rates were
18% and 12% of that found for 2AM. Focusing on the more
promiscuous Ppo enzyme, we found that ethanolamine was
Bioinformatic sequence analyses. Comparative analyses
of protein amino acid sequences used pBLAST from the
NCBI website. Alignments were determined by ClustalW
multiple-sequence alignment [16].

Medium-Chain Dehydrogenases with New Specificity
Protein & Peptide Letters, 2014, Vol. 21, No. 1 13
Table 1. A comparison of relative velocities for different sub-
strates versus dehydrogenases from B. amyloliquefa-
ciens and P. polymyxa. Velocities in each column are
expressed as percentages relative to the velocity with
2AM as substrate. ND = not determined. Conditions
are provided in Materials and methods.
high 10 mM) may fail to reveal activity with very weakly
bound substrates, it appears that the enzyme from P. po-
lymyxa is more tolerant of structural diversity than that from
B. amyloliquefaciens, and 2AM is the preferred substrate.
This latter result drives home the point that 2AM is a key
intermediate in azasugar biosynthesis, as shown previously
[9]. The slow turnover of the enzyme is understandable
given its role in secondary metabolism, [19] and the lack of
strong evolutionary pressure to maintain the pathway as evi-
denced by the lack of azasugar production in B. subtilis de-
spite its otherwise close relationship to the select members of
the genus that are known azasugar producers. The polyol
dehydrogenases (EC 1.1.1.14) as exemplified by “GutB”
dehydrogenase from B. subtilis are so-named for their ability
to oxidize polyol substrates such as sorbitol, xylitol and L-
iditol [18]. Despite sequence similarity of the Bam and Ppo
enzymes studied here to B. subtilis GutB, (47% and 46%
similarity at the protein level) they disdain uncharged polyol
substrates, but rather prefer amino-substituted polyols. These
enzymes are members of the broad super family of medium
chain dehydrogenases, and to the best of our knowledge, as
aminopolyol dehydrogenases, they are a unique addition to
the MDR superfamily. Based on the specificity we have ob-
served we term these enzymes as amino-mannitol dehydro-
genases (AMDHs).
relative activity
relative activity
substrates
(Bam)
(Ppo)
fructose
mannitol
0%
9%
0%
0%
0%
0%
ND
ND
ND
ND
ND
ND
ND
3%
0%
0%
18%
12%
0%
3%
0%
0%
0%
0%
mannose
sorbitol
4-amino-1-butanol
6-amino-1-hexanol
serinol
L-threonine
glucosamine
ethanolamine
phenylpropanolamine
dimethylethanolamine
Clues as to the origin of the specificity for amino polyol
substrates were found in sequence analysis and the structure
of sheep liver sorbitol dehydrogenase [20].
Figure 3 presents a ribbon diagram for sorbitol dehydro-
genase with a glycerol bound in the active site as a binary
complex. Arginine 297 is in hydrogen bonding contact with
the bound glycerol. Sequence alignments (Figure 3, inset) of
sheep and Bacillus subtilis sorbitol dehydrogenases versus
bacterial dehydrogenases from known or putative azasugar
producers reveal that the position homologous to 297 is sub-
stituted with either D or E for azasugar producers versus R.
Possibly, D or E residues provide charge recognition with
the positively charged ammonium group of an amino polyol
not a substrate, indicating that a carbon chain length of two
was too short. L-threonine was a very poor substrate distin-
guishing this enzyme from the threonine dehydrogenases that
are also members of the MDR superfamily. We also tested
glucosamine, phenylpropanolamine, and dimethyl ethanola-
mine versus the Ppo enzyme and found that none of these
substrates were accepted. Keeping in mind the caveat that
screening at fixed substrate concentration (albeit a relatively
Figure 3. Ribbon diagram for sorbitol dehydrogenase from sheep liver (PDB ID 3EQ3). Arginine 297 is represented with space-filling atoms
as is bound glycerol. The inset presents CLUSTAL sequence alignment of putative aminopolyol dehydrogenases from azasugar producers
and sorbitol dehydrogenases from sheep liver and Bacillus subtilis. The black square indicates position 297 for sheep liver sorbitol dehydro-
genase.

14 Protein & Peptide Letters, 2014, Vol. 21, No. 1
Wu et al.
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mutant (ꢀrel = 0.43). The preference for the charged 2AM
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porting the proposed role for D283 in aminopolyol recogni-
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CONCLUSION
The NAD-dependent dehydrogenases that appear in
azasugar biosynthetic clusters are members of the medium
chain reductase/dehydrogenase family that have specificity
for 2-amino-2-deoxy-D-mannitol, but in some cases are suf-
ficiently promiscuous to accept simple amino alcohols as
substrate. In codon optimized form these enzymes are well-
expressed in E. coli, and may be a useful platform for evolu-
tion of redox catalysts that accept synthetically interesting
precursors. Continuing studies are aimed at further defining
the basis for molecular recognition within the active site via
structural studies.
[12]
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CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flicts of interest.
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ACKNOWLEDGEMENT
We are grateful for the support of the National Science
Foundation (MCB-1020940).
SUPPLEMENTARY MATERIAL
[16]
Supplementary material is available on the publishers
Web site along with the published article.
[17]
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Received: May 31, 2013
Revised: August 4, 2013
Accepted: August 4, 2013
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