Producing Glucose 6-phosphate from cellulosic biomass: Structural insights into levoglucosan bioconversion

Article abstract of DOI:10.1074/jbc.M115.674614

The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1, 6-anhydro-β-D-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1, 6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom. Using x-ray crystallography, we show that LGK binds two magnesium ions in the active site that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. To further verify the metal binding sites, we co-crystallized LGK in the presence of manganese instead of magnesium and solved the structure de novo using the anomalous signal from four manganese atoms in the dimeric structure. The first metal is required for catalysis, whereas our work suggests that the second is either required or significantly promotes the catalytic rate. Although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1, 6-anhydro-N-acetylmuramic acid kinase (AnmK), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. We also observed a second alternate binding orientation for levoglucosan, and in these structures, ADP was found to bind with lower affinity. These combined observations provide an explanation for the high K_m of LGK for levoglucosan. Greater knowledge of the factors that contribute to the catalytic efficiency of LGK can be used to improve applications of this enzyme for levoglucosan-derived biofuel production.

Full text of DOI:10.1074/jbc.M115.674614

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 44, pp. 26638–26648, October 30, 2015
Published in the U.S.A.
Producing Glucose 6-Phosphate from Cellulosic Biomass
STRUCTURAL INSIGHTS INTO LEVOGLUCOSAN BIOCONVERSION*
Received for publication, June 23, 2015, and in revised form, September 4, 2015 Published, JBC Papers in Press, September 9, 2015, DOI 10.1074/jbc.M115.674614
John-Paul Bacik‡1, X Justin R. Klesmith§, Timothy A. Whitehead¶ʈ, Laura R. Jarboe**, Clifford J. Unkefer‡,
Brian L. Mark‡‡, and Ryszard Michalczyk‡
From the ‡Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, the Departments of §Biochemistry
and Molecular Biology, ¶Chemical Engineering and Materials Science, and ʈBiosystems and Agricultural Engineering, Michigan
State University, East Lansing, Michigan 48824, the **Department of Chemical and Biological Engineering, Iowa State University,
Ames, Iowa 50011, and the ‡‡Department of Microbiology, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada
Background: Levoglucosan kinase converts the cellulosic biomass pyrolysis product levoglucosan to the common metab-
olite glucose 6-phosphate.
Results: Crystallographic structures demonstrate two metal binding sites and an alternate binding arrangement for
levoglucosan.
Conclusion: The unusual mode of levoglucosan binding provides a rationale for the high Km of LGK for its substrate.
Significance: These results provide a greater understanding of the underlying biochemistry of anhydrosugar processing.
The most abundant carbohydrate product of cellulosic bio- the factors that contribute to the catalytic efficiency of LGK can
mass pyrolysis is the anhydrosugar levoglucosan (1,6-anhydro- be used to improve applications of this enzyme for levogluco-
␤-D-glucopyranose), which can be converted to glucose 6-phos- san-derived biofuel production.
phate by levoglucosan kinase (LGK). In addition to the canonical
kinase phosphotransfer reaction, the conversion requires cleav-
age of the 1,6-anhydro ring to allow ATP-dependent phosphor-
ylation of the sugar O6 atom. Using x-ray crystallography, we
show that LGK binds two magnesium ions in the active site that
are additionally coordinated with the nucleotide and water mol-
ecules to result in ideal octahedral coordination. To further ver-
ify the metal binding sites, we co-crystallized LGK in the pres-
ence of manganese instead of magnesium and solved the
structure de novo using the anomalous signal from four manga-
nese atoms in the dimeric structure. The first metal is required
for catalysis, whereas our work suggests that the second is either
required or significantly promotes the catalytic rate. Although
the enzyme binds its sugar substrate in a similar orientation
to the structurally related 1,6-anhydro-N-acetylmuramic acid
kinase (AnmK), it forms markedly fewer bonding interactions
with the substrate. In this orientation, the sugar is in an optimal
position to couple phosphorylation with ring cleavage. We also
observed a second alternate binding orientation for levogluco-
san, and in these structures, ADP was found to bind with lower
affinity. These combined observations provide an explanation
for the high Km of LGK for levoglucosan. Greater knowledge of
The anhydrosugar levoglucosan (1,6-anhydro-␤-D-glucopy-
ranose) is a well recognized product of biomass burning that is
often used as a molecular tracer of such events in soils, aerosols,
snow pits, and even human urine (1–6). Although not as abun-
dant as levoglucosan, other anhydrosugars such as mannosan,
galactosan, levogalactosan, levomannosan, and cellobiosan
have also been detected in aerosol and soil samples (1–4). It has
been estimated that 4 billion metric tons of carbon are released
globally by biomass burning every year (7). A characterization
of wildfire smoke demonstrated that anhydrosugars were the
second most abundant form of organic carbon in given samples,
at a concentration of 24 mg/g of organic carbon (8), leading to
an estimated annual production of 90 million metric tons of
anhydrosugars. These anhydrosugars thus represent a signifi-
cant yet undercharacterized portion of the global carbon cycle.
Although the radical-mediated degradation of aerosol-associ-
ated levoglucosan has been observed (9, 10), it has also been
reported that levoglucosan, and presumably other anhydrosug-
ars, can return to the soil via rainwater (11). Once in the soil,
these sugars are subjected to further degradation (12). Two dis-
tinct biological pathways have been identified during levoglu-
cosan degradation. The ATP-dependent levoglucosan kinase
(LGK)2 pathway (Fig. 1), which is present in some fungi, pro-
duces glucose 6-phosphate (G6P), a common metabolic inter-
mediate (13). The modularity of this pathway has been demon-
strated by its functional expression in bacteria such as
* This work was supported in part by the Natural Sciences and Engineering
Research Council of Canada (NSERC) (Grant 311775-2010) (to B. L. M.), a
Manitoba Research Chair (to B. L. M.), and a Manitoba Health Research
Council Postdoctoral Fellowship (to J. P. B.). This work was also partially
funded through the Protein Crystallography Station from the Department
of Energy Office of Biological and Environmental Research (to J. P. B. and
C. J. U.) and National Science Foundation (NSF) CAREER Award 1254238 (to
J. R. K. andT. A. W.). Theauthorsdeclarethattheyhavenoconflictsofinter- 2 The abbreviations used are: LGK, levoglucosan kinase; LG, levoglucosan;
est with the contents of this article.
G6P, glucose 6-phosphate; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)-
ethyl]amino}-1-propanesulfonic acid; TCEP, tris(2-carboxyethyl)phosphine;
Bis-Tris, 2-(bis(2-hydroxyethyl)amino)-2-(hydroxymethyl)propane-1,3-diol;
Bis-Trispropane, 1,3-bis[tris(hydroxymethyl)methylamino]propane;AMPPCP,
adenosine 5Ј-(␤,␥-methylene)triphosphate; anhMurNAc, 1,6-anhydro-N-
acetylmuramic acid; AnmK, 1,6-anhydro-N-acetylmuramic acid kinase.
The atomic coordinates and structure factors (codes 4ZFV, 4YH5, 4ZLU, 5BVC,
and 5BSB) have been deposited in the Protein Data Bank (http://wwpdb.org/).
1 To whom correspondence should be addressed: Bioscience Division, Los
Alamos National Laboratory, TA03, Bldg. 4200, MS T007, Los Alamos, NM
87545. Tel.: 505-667-3221; E-mail: jbacik@lanl.gov.
26638 JOURNAL OF BIOLOGICAL CHEMISTRY
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Structural Insights into Levoglucosan Bioconversion
kinases in the hexokinase family (23). They share significant
sequence homology and a similar fold and domain architecture,
including a nucleotide binding domain and a sugar binding
domain that is separated by a dynamic hinge region (23). We
previously determined the crystal structures of AnmK from
Pseudomonas aeruginosa bound to its sugar substrate and a
product ADP (24). An aspartate residue (Asp-182) was identi-
fied as the general base that initiates the attack of a nucleophilic
water molecule on the anomeric carbon (C1) of anhMurNAc,
thereby promoting cleavage of the 1,6-anhydro bond and trans-
fer of the ␥-phosphate of ATP to the O6 oxygen of the sugar.
Subsequent structural studies of AnmK in the open conforma-
tion bound to the ATP analog, AMPPCP, provided the basis for
the conformational dynamics of the enzyme during its catalytic
cycle, whereby it cycles between a catalytically competent
closed state and an open state (25).
LGK was first isolated from fungal sources and was subse-
quently determined to require ATP and magnesium for phos-
phorylation of levoglucosan (13). From the perspective of met-
abolic engineering, levoglucosan is not within the native
substrate range of common industrial biocatalysts. Recently,
codon-optimized LGK from Lipomyces starkeyi was introduced
into ethanologenic E. coli by chromosome integration. The
FIGURE 1. Conversion of cellulose to levoglucosan through pyrolysis is
followed by enzymatic cleavage and phosphorylation through the
action of levoglucosan kinase to produce G6P.
Escherichia coli (14). A bacterial levoglucosan dehydrogenase engineered strain was found to be able to use levoglucosan as
pathway that converts levoglucosan to 3-keto-levoglucosan in the only carbon source for ethanol production (14). However,
ϩ
an NAD -dependent reaction has also been described (15). LGK enzymes display a relatively low binding affinity for levoglu-
However, the detailed mechanisms of LGK and levoglucosan cosan (Km ϭ 69–105 mM (26, 27)), consistent with the residual
dehydrogenase, associated metabolic pathways, and the iden- levoglucosan left unutilized during fermentation (14). Although it
tity of levoglucosan membrane transporters remain unknown. was discovered almost 25 years ago and clearly has potential as a
An increased understanding of the biological pathways associ- catalyst in advanced biofuels production, relatively little is known
ated with the microbial degradation of these sugars can be real- about LGK. Crystallographic structures of L. starkeyi LGK, the
ized by identifying and characterizing the enzymes and cofac- object of this study, provide detailed insights in to the mechanism
tors required for these specific reactions.
of LGK, including binding of two divalent metal ions and an
Beyond revealing the fundamental science of anhydrosugar unusual binding mode of levoglucosan.
metabolism, the potential applications of these sugars are inter-
Experimental Procedures
esting from the perspective of bioengineering. Biomass is an
attractive source of carbon and energy for the production of
LGK Expression and Purification—The codon optimized lgk
renewable biofuels and chemicals. The use of corn- and sugar gene from L. starkeyi YZ-215 (GenBank accession number
cane-derived glucose for fermentative production of ethanol is EU751287) was originally synthesized by GenScript (Piscat-
an example of this biomass-to-biofuel pathway. The use of away, NJ) as described (14). The gene was then PCR-amplified
lignocellulosic biomass, such as switchgrass or crop residues, from its pUC57 construct using Phusion DNA polymerase
shows several advantages when compared with these feedstocks (New England Biolabs) and oligonucleotide primers 5Ј-
due to issues related to food/fuel conflicts, land usage and eco- GATATACATATGCCGATTGCGACGTCTACCG-3Ј and
nomic viability (16, 17). With the inherent recalcitrance of ligno- 5Ј-GATATAGGATCCCGCCCAGTTGTTGGTGATAAC-3Ј
cellulosic biomass to efficient depolymerization into clean, fer- (Alpha DNA, Montreal, Quebec, Canada). The PCR amplicon
mentable sugars (18, 19), it is interesting to consider that and a modified version of a pET28 plasmid (Novagen) contain-
thermochemical processing of such biomass by fast pyrolysis ing a reduced multicloning site (NdeI/SpeI/BamHI) and a
results in the recovery of a significant portion of biomass-associ- C-terminal His6 tag were restricted with NdeI and BamHI and
ated sugars as anhydrosugars (20). Knowledge of the existing bio- ligated using T4 DNA ligase (New England Biolabs). The liga-
logical pathways related to these sugars must be expanded if they tion product, recombinant plasmid pLSlgk, was then used to
are to be used effectively as carbon and energy sources for the transform E. coli strain BL21(DE3) GOLD cells (Stratagene) for
fermentative production of biorenewable fuels and chemicals.
Anhydrosugars are also produced naturally during depoly-
further expression and purification.
Recombinant E. coli BL21(DE3) GOLD cells harboring
merization of the bacterial cell wall for the purpose of pepti- pLSlgk were grown to an A600 of ϳ 0.5 at 37 °C, with shaking, in
doglycan recycling (21). The resulting 1,6-anhMurNAc sugar is 500-ml volumes of LB medium supplemented with 35 ␮g/ml
cleaved and phosphorylated by AnmK, which is expressed by kanamycin. Expression of LGK was induced with 1 mM isopro-
many Gram-negative bacteria, to produce MurNAc-6-phos- pyl-1-thio-␤-D-galactopyranoside for 3 h at 30 °C, with shaking.
phate (22). LGK and AnmK form a sub-family of anhydrosugar Cells were pelleted by centrifugation and stored at Ϫ80 °C. Pel-
OCTOBER 30, 2015•VOLUME 290•NUMBER 44
JOURNAL OF BIOLOGICAL CHEMISTRY 26639

Structural Insights into Levoglucosan Bioconversion
lets were thawed in 20 ml of ice-cold lysis buffer (0.5 M NaCl, 20 the monomeric structure with levoglucosan in the alternate
mM Tris-HCl, pH 7.5, 0.1 mM PMSF, 2 mM imidazole) and lysed orientation, were collected at beamline 08ID-1 at the Canadian
using a French pressure cell press (Ameco). The lysate was clar- Light Source (Saskatoon, Saskatchewan, Canada), integrated
ified by centrifugation and mixed with 2 ml of TALON metal using XDS (28), and scaled and merged using SCALA (29). For
affinity resin (Clontech) with gentle shaking for 30 min at room the LGK structure bound to manganese, a single-wavelength
temperature. The TALON beads were centrifuged and re-sus- anomalous diffraction dataset was collected (␭ ϭ 1.8920;
pended in binding buffer (500 mM NaCl, 20 mM Tris, pH 7.5, 0.5 energy ϭ 6.55306; fЉ ϭ 8.42; fЈ ϭ Ϫ7.46). The monomeric struc-
mM TCEP) before being poured into a 20-ml gravity column. ture with levoglucosan bound in the alternate orientation was
The column was washed with 20 ml of binding buffer supple- collected at the Stanford Synchrotron Radiation Lightsource
mented with 5 mM imidazole (Sigma), followed by 20 ml of beamline BL7-1, integrated using MOSFLM (30), and scaled
binding buffer supplemented with 10 mM imidazole. The LGK and merged using SCALA.
protein was eluted from the column with 10 ml of binding
Phase estimates for the structure bound to magnesium were
buffer supplemented with 250 mM imidazole. The eluate was initially obtained by molecular replacement using PHASER (31)
dialyzed overnight against 20 mM Tris, pH 7.5, 100 mM NaCl, 1 and the structure of AnmK from P. aeruginosa as a search
mM EDTA, 5 mM DTT. The protein was further purified by gel model (PDB identifier: 3QBW). The model was subsequently
filtration (Superdex 200) in 20 mM Tris, pH 7.5, 50 mM NaCl, 0.5 rebuilt using the PHENIX autobuild routine (32) for initial
mM TCEP prior to concentration using an Amicon Ultra-15 structural analysis. For the structure bound to manganese,
concentrator with a 10,000 Da cut-off (Millipore). Chromato- HySS (33) was used to locate four manganese atoms (1.9 Å
graphic steps were performed using an ÄKTA FPLC (GE resolution cut-off used, anomalous signal ϭ 0.0781; occupan-
Healthcare).
cies ϭ 1.00, 0.96, 0.96, 0.94). Phasing was done using
LGK Crystallization, Data Collection, and Structure PHASER-EP (final figure of merit ϭ 0.39) prior to density mod-
Determination—LGK crystals were grown at room tempera- ification using the resolve routine in AUTOSOL, and the out-
ture using the hanging drop vapor diffusion method by mixing put density modified map file was used as an initial map file for
equal volumes of reservoir buffer (22% PEG 4000, 100 mM subsequent automated model building using the PHENIX auto-
sodium acetate, 100 mM Tris, pH 7.5) and LGK (7 mg/ml) in build routine (32), which successfully built 862 of 894 protein
magnesium crystallization buffer (50 mM NaCl, 2 mM ADP, 4 residues (correlation coefficient ϭ 0.84). This structure was
mM MgCl2, 0.5 mM TCEP, 20 mM Tris, pH 7.5) for the structure then subsequently used as the starting model for the structure
bound to magnesium. For the structure bound to manganese, bound to magnesium and ADP to reduce model bias with the
equal volumes of reservoir buffer (18% PEG 4000, 100 mM previously built model using the AnmK structure for molecular
sodium acetate, 100 mM Tris, pH 7.2) and LGK (7 mg/ml) in a replacement. The remainder of the LGK structures were built
manganese crystallization buffer (50 mM NaCl, 2 mM ADP, 5 using the experimentally determined LGK structure as a start-
mM manganese(II) chloride tetrahydrate, 25 mM levoglucosan, ing model, with rounds of iterative model building and refine-
0.5 mM TCEP, 20 mM Tris, pH 7.5) were mixed. For the struc- ment performed using Coot and PHENIX (34). The stereo-
ture bound to magnesium, ADP, and levoglucosan, equal vol- chemical quality of the final models was assessed using
umes of reservoir buffer (19% PEG 4000, 100 mM sodium ace- MolProbity (35). Refinement statistics are presented in Table 1.
tate, 100 mM Tris, pH 6.8) and LGK (7 mg/ml) in crystallization All structural figures were prepared using PyMOL (36).
buffer (50 mM NaCl, 2 mM ADP, 4 mM MgCl2, 0.5 mM TCEP, 1
Enzyme Kinetics—LGK activity assays were performed using
mM aluminum nitrate, 10 mM sodium fluoride, 20 mM Tris, pH the G6P dehydrogenase coupling system (37). For the reaction
7.5) were mixed, and a resultant crystal was then soaked directly velocity as a function of ATP concentration, all assays were
in the crystallization drop with 100 mM levoglucosan for 10 carried out in at least triplicate in a buffered solution of 4 or 8
ϩ
min. The monomeric structure bound to levoglucosan in the mM MgCl2, 50 mM TAPS, pH 9.0, 1 mM NADP , 1 unit of G6P
productive conformation was determined by mixing equal dehydrogenase from Saccharomyces cerevisiae (Sigma), and 2.4
amounts of LGK (9 mg/ml) in crystallization buffer (50 mM ␮g of LGK. The reaction was initiated by the addition of levo-
NaCl, 0.5 mM TCEP, 20 mM Tris, pH 7.5, 200 mM levoglucosan) glucosan (Carbosynth) at a concentration of 100 mM, bringing
and reservoir buffer (1.8 M ammonium sulfate, Hepes 7.0, and the final volume to 1 ml in a quartz reaction vessel. The change
100 mM sodium acetate). The monomeric structure bound to in A340 over a 2-min time period at 30 °C was measured using a
ADP and levoglucosan (alternate orientation only) was deter- Cary 300 spectrophotometer equipped with a Peltier tempera-
mined by mixing equal amounts of LGK (25 mg/ml) in crystal- ture controller. The graph was plotted using Prism 6 software
lization buffer (100 mM NaCl, 0.5 mM TCEP, 20 mM Tris, pH (GraphPad Software, La Jolla, CA). LGK reaction velocity as a
7.5, 2 mM ADP, 4 mM MgCl2, 1 mM aluminum nitrate, and 10 function of levoglucosan concentration and buffer type was
mM sodium fluoride) and reservoir buffer (1.3 M sodium malo- assayed using 100-␮l microplates in the presence of HEPES and
nate, 93 mM Bis-Tris propane, pH 7.0) and then soaking a Tris at pH 7.6 at a concentration of 55 mM. All assays were
ϩ
resultant crystal in the drop with 200 mM levoglucosan. Crystals performed with 99 mM NaCl, 1.5 mM NAD , 2 mM ATP, 20 mM
were cryoprotected by dragging them through a drop contain- MgCl2, and 0.8 units of G6P from Leuconostoc mesenteroides
ing cryoprotectant solution (reservoir buffer supplemented (Sigma). Levoglucosan was added to each assay well in a 1:2
with 9% sucrose (w/v), 2% glucose (w/v), 8% glycerol (v/v), and serial dilution at final concentrations ranging from 550 to 17.2
8% ethylene glycol (v/v)) prior to being flash-cooled in liquid mM. Assay components were sealed and incubated on a 30 °C
nitrogen. X-ray diffraction data for all of the structures, except pebble bath for 5 min prior to the addition of 10 ␮l of purified
26640 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 290•NUMBER 44•OCTOBER 30, 2015

Structural Insights into Levoglucosan Bioconversion
TABLE 1
Crystallographic data processing and refinement statistics for levoglucosan kinase crystallographic structures
PDB ID (ligand)
5BVC (ADP/Mg/LG
(alternate))
4ZFV (ADP/Mg)
4YH5 (ADP/Mn)
4ZLU (ADP/Mg/LG)
5BSB (LG)
Data collectiona
Space group
P41212
P41212
P41212
P41212
P41212
Unit cell (Å)
a ϭ b ϭ 114.40,
c ϭ 232.50
a ϭ b ϭ 114.44,
c ϭ 232.48
a ϭ b ϭ 114.86,
c ϭ 234.77
a ϭ b ϭ 70.25,
c ϭ 264.19
a ϭ b ϭ 70.32,
c ϭ 263.33
␣ ϭ ␤ ϭ ␥ ϭ 90.00
0.9795
␣ ϭ ␤ ϭ ␥ ϭ 90.00
1.8920
␣ ϭ ␤ ϭ ␥ ϭ 90.00
0.9795
␣ ϭ ␤ ϭ ␥ ϭ 90.00
1.1271
␣ ϭ ␤ ϭ ␥ ϭ 90.00
0.9795
Wavelength (Å)
Resolution range (Å)
Total observations
Total unique observations
47.20–1.50 (1.58–1.50) 47.21–1.90 (2.00–1.90) 47.57–1.80 (1.90–1.80) 43.27–2.00 (2.11–2.00) 48.86–1.85 (1.95–1.85)
1,365,581
244,767
2,394,174
121,746
567,803
148,841
325,310
145,247
44,939
57,761
I/␴
10.3 (2.1)
21.6 (4.2)
9.1 (2.2)
6.2 (2.0)
9.8 (1.9)
I
Completeness (%)
Rmerge
99.9 (100.0)
0.099 (0.787)
0.045 (0.363)
5.6 (5.5)
99.9 (99.1)
0.104 (0.569)
0.024 (0.175)
19.7 (11.2)
99.8 (98.5)
10.2 (5.6)
99.9 (99.7)
0.101 (0.719)
0.059 (0.397)
3.9 (4.2)
98.5 (99.9)
0.100 (0.592)
0.062 (0.368)
3.3 (3.4)
100.0 (100.0)
0.096 (0.861)
0.043 (0.395)
5.6 (5.7)
Rpim
Redundancy
Anomalous completeness (%)
Anomalous multiplicity
Refinement statistics
Resolution (Å)
47.20–1.50
244,650
12,339
47.21–1.90
121,618
6081
47.10–1.80
145,033
7286
43.3–2.00
44,796
48.86–1.85
57,602
Reflections (total)
Reflections (test)
Total atoms refined
Solvent
2271
2897
8309
7824
7762
3610
3680
1440
1032
1001
292
359
R
work (Rfree
)
0.16 (0.17)
0.007/1.095
0.16 (0.17)
0.007/1.069
98.3/1.5
0.19 (0.22)
0.008/1.145
98.3/1.5
0.20 (0.23)
0.008/1.117
97.7/2.1
0.19 (0.22)
0.006/1.035
97.2/2.6
RMSDsb (bond lengths (Å)/angles (°))
Ramachandran plot (favored/allowed (%)) 98.1/1.7
Mean B values (Å2)
Overall protein chain (A/B)
15.7/17.0
20.1/21.6
20.4/27.2
21.5/74.9
23.8, 20.8
30.3/25.5
28.0
50.6
42.0
20.8
29.6
ADP
13.0/15.5
18.3/20.6
Mg or Mn
LG
12.6,12.4/13.6,13.7
27.3,28.3/29.2,29.5
26.4
Active site Tris
26.9
a Values in parentheses refer to the high-resolution shell.
b RMSDs, root mean square deviations.
LGK for an assay enzyme concentration of 0.1 ␮M. A340 was the presence of ADP and magnesium, and the structure was
monitored over a 6-min time period by a Synergy H1 spectro-
photometer (BioTek, Winooski, VT) using a kinetic read
method every 21 s at a controlled temperature of 30 °C. Prism 6
software was used to calculate the Michaelis-Menten parame-
ters using a non-linear fit of the datasets.
solved to 1.5 Å in the tetragonal space group P41212 as a non-
crystallographic dimer (Fig. 2A; Table 1). The structure was
initially solved using the closed AnmK structure (3QBW) as the
molecular replacement search model. Each monomer of LGK
contains a deep hinge between the two domains where the sub-
strates bind. In many hexokinases, including AnmK, these two
domains have often been shown to rotate apart from one
another (25), whereas in this structure, the domains appear to
be closed. Although longer in amino acid sequence, the overall
tertiary structure of LGK is similar to the structure of AnmK in
the closed conformation (root mean square deviation of C-al-
phas ϭ 1.8 Å), including a conserved dimeric interface. LGK is
somewhat larger than AnmK, with extra helical and loop
arrangements at the bottom of the hinge region (Fig. 2A).
LGK binds the reaction product ADP through multiple
hydrogen bonds (Fig. 2B), including bonds between the adenyl
moiety and Asp-237, between the nucleotide ribose and Asp-
221, and between the ADP phosphates and protein residues
Ser-24, Gly-189, and Gly-328. Similarly to the vast majority of
other kinases, magnesium is required for LGK catalysis (13). A
striking observation found in the electron density map for the
LGK structures is the apparent binding of two magnesium ions
in the active site. The magnesium ions are observed in ideal
octahedral coordination with Glu-362 and Asp-26, several
water molecules, and the ADP (Fig. 2B). The first of the bound
metals, designated M1, forms an electrostatic interaction with
the ␤-phosphate, and its positioning suggests that it plays a
direct role in phosphoryl transfer. The second of these metals,
1H NMR Studies—All spectra were recorded on a 700-MHz
Bruker AVANCE III instrument equipped with a TCI cryo-
probe at 20 °C. 38 ␮l of enzyme solution was added to a total
reaction volume of 600 ␮l of solution containing all other re-
agents (20 mM levoglucosan, 4.5–7 units of lactic dehydroge-
nase, 3–5 units of pyruvate kinase from rabbit muscle (Sigma),
15 mM phosphoenolpyruvic acid, 1 mM ATP, 10 mM magne-
sium, 60 mM NaCl, and 100 mM Bis-Tris, pD 6.9). The sample
was mixed, the time of mixing was recorded, and the sample
was placed on the magnet. Spectra were recorded at predefined
intervals with a single scan recorded for each spectrum to
obtain the exact time stamp and to avoid partial saturation of
resonances due to insufficient relaxation delay. Spectra were
processed using the MNova 9.1 suite (Mestrelab Research), and
intensities were determined from spectral deconvolution.
Buffer resonance at 1.88 ppm was also quantified to ensure
consistency of measurements. Intensities were fitted to a single
exponential growth/decay function, and plots were generated
using Origin 7 software.
Results
Overall Structures, Metal Binding, and Active Site Co-
ordination—L. starkeyi LGK was targeted for crystallographic
studies to gain a better understanding of its structure, mecha-
nism, and substrate interactions. LGK was first crystallized in designated M2, likely plays a role in coordinating the position of
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Structural Insights into Levoglucosan Bioconversion
A
B
Asp221
LGK
Asp237
Tyr234
ADP
Ser24
Gly189
Asp26
Gly328
Mg2+
(M2)
Mg2+
(M1)
Tyr331
Glu362
Chain A
Chain B
C
Asp221
AnmK
Asp237
Tyr234
ADP
Ser24
Gly189
Asp26
Gly328
Mn2+
Mn2+
Chain A
Tyr331
Glu362
Chain B
D
E
F
ADP
4 mM Mg
8 mM Mg
M2
M1
Asp212
Tris
FIGURE 2. A, crystal structures of LGK (top) and AnmK (bottom) bound to ADP. B, the nucleotide binding site demonstrates octahedral coordination of two magnesium
atoms. Residues involved in binding are shown in stick format with carbons in cyan, nitrogens in blue, and oxygens in red. For the ADP, the carbon and phosphorous
atoms are shown in yellow and orange, respectively. Waters and magnesiums are shown as red and green spheres, respectively. The gray mesh represents the 2mFo Ϫ
DFc electron density map contoured around the ADP and magnesiums at 1␴. Molecular bonds are shown as yellow dashed lines. C, the nucleotide binding site in the
presence of ADP and manganese. Anomalous difference Fourier map contoured around the manganese atoms is shown as a purple mesh at 3␴, while the manganese
atoms are shown as purple spheres. D, LGK reaction velocity as a function of varying ATP concentrations and two constant magnesium concentrations. The vertical
dotted lines delineate the concentration at which ATP and magnesium are equal. Error bars indicate means ϮS.E. E, a molecule of Tris bound in the LGK active site. The
mFo Ϫ DFc omit map is shown as a green mesh contoured at 2.5␴. F, Lineweaver-Burk plots of LGK reaction.
the ␣- and ␤-phosphates because it binds to both of these phos- catalysis when ATP concentrations are less than the magne-
phates, whereas a role in modulation of electrostatic charges is sium concentrations, whereas free magnesium would be
also plausible.
depleted when ATP concentrations exceeded the magnesium
The binding of two divalent metal ions by LGK was further concentration. The resulting data are shown in Fig. 2D, with a
verified by co-crystallizing the enzyme with manganese instead dramatic drop-off in activity when the ATP concentration
of magnesium and solving the structure de novo using the exceeds the magnesium concentration at both 4 mM and 8 mM
anomalous signal of the four manganese atoms present in the magnesium. Given this result and our structural observations,
dimeric structure (Fig. 2C). The initial molecular replacement we favor a model whereby either both magnesiums are required
solution for the LGK structure bound to ADP and magnesium for catalysis or the second magnesium greatly enhances the rate
was subsequently rebuilt using the experimentally determined of activity. An alternate explanation for the observed rates of
LGK structure as a starting model to reduce phase error and activity could be that free ATP is acting as an inhibitor of the
model bias from the AnmK structure. Although magnesium reaction when free magnesium is depleted, although our struc-
has previously been shown to be required for the LGK reaction, tural work shows that the nucleotide binds much more strongly
we sought to determine whether both magnesiums are required when magnesium is also bound (see below). It should also be
for catalysis. Considering a dissociation constant (KD) of 50 ␮M considered that although both metals play a role in catalysis, the
for the MgATP complex (38), we analyzed the LGK reaction use of two magnesiums may also limit the rate of ADP product
rate holding the magnesium concentration at two constant val- release following catalysis, and further kinetic studies will be
ues (4 or 8 mM) and varying the ATP concentrations. Using this required to more thoroughly investigate the kinetic conse-
reasoning, a second magnesium atom would be available for quences of two magnesium binding sites.
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Structural Insights into Levoglucosan Bioconversion
A
B
C
ADP
ADP
Leu142
Val143
Arg129
Thr116
Asp212
M2
Wat
A
M1
LG
anhMurNAc
Thr97
Asp182
Asp212
Asp182
3.8
2.6
4.0
2.8
Wat
Wat
A
A
E
D
FIGURE 3. A, LGK active site in the presence of ADP, magnesium, and levoglucosan. Black dashed lines indicate distances in angstroms between moieties that
are important for catalysis. The green mesh represents an mFo Ϫ DFc omit map calculated after the removal of ADP, magnesium, and levoglucosan shown at
2.5␴. WatA indicates water molecule. B, model of AnmK bound to ADP and anhMurNAc. The structure was modeled by superposing the ADP-bound crystal-
lographic structure onto the anhMurNAc-bound crystallographic structure. C, superposition of the LGK and AnmK sugar binding sites. Protein carbon atoms of
residues involved in binding of the sugars are shown in gray and cyan for LGK and AnmK, respectively. Carbon atoms for the sugar are shown in light green
(anhMurNAc) or green (levoglucosan). D, 1H NMR analysis of the LGK reaction. Peaks corresponding to the anomeric protons of the substrate (levoglucosan,
5.31 ppm) and products (␣ and ␤ anomers of G6P) are labeled. Peaks due to the presence of phosphoenolpyruvic acid (PEP) are also labeled. e, rates of
levoglucosan consumption and G6P formation measured using 1H NMR are shown over time.
Interestingly, a molecule of Tris, which was present in the positioning of levoglucosan in the A-chain monomer is very
crystallization buffer, was also observed bound in the active site similar to the anhMurNAc in the binding site of AnmK (Fig. 3,
in one of the LGK monomers bound to magnesium and ADP A–C). LGK exhibits a very high Km for its substrate when com-
(Fig. 2E). Because the Tris molecule forms a hydrogen bond pared with AnmK (0.2 mM) and, accordingly, forms fewer
with the catalytic aspartate (Asp-212), it occupies roughly the bonding interactions (24). Levoglucosan forms only a single
same position where it is expected that levoglucosan would be hydrogen bond with LGK (Asp-212), although the substrate
coordinated. Interestingly, analysis of the Lineweaver-Burk also appears to be stabilized by hydrogen bonds with water
plots of the reaction in the presence of either Tris or HEPES as molecules in the active site. Specifically, a water molecule
the buffer demonstrated that Tris is a competitive inhibitor of appears to mediate a hydrogen-bonding network between Thr-
the reaction (Fig. 2F). We determined a Km of 119 Ϯ 12 mM for 116 and O1 of LG (Fig. 3C). In AnmK, a similar bonding
levoglucosan in HEPES buffer and an apparent Km of 255 Ϯ 20 arrangement is observed between the analogous threonine res-
mM in Tris-HCl buffer, resulting in a KI for Tris of 48 Ϯ 27 mM. idue and the acetamido group of anhMurNAc, suggesting that
Tris has been shown to inhibit the catalytic activity of other the threonine residue plays a conserved role in substrate bind-
enzymes (39–41), and although its KI appears relatively high, ing. Although aluminum nitrate and sodium fluoride were also
the comparably high Km of LGK for levoglucosan suggests that present in the crystallization buffer, the expected aluminum
the inhibition is significant.
fluoride could not be unambiguously modeled in the resulting
Mechanism of Sugar Cleavage—To gain insights into the data. However, the ADP and two magnesium ions in this mono-
mechanism of 1,6-anhydro bond cleavage and to evaluate sugar mer (Fig. 3A) are observed with similar bonding arrangements
binding interactions including a possible rationale for the high to those shown in Fig. 2.
intrinsic Km of LGK for levoglucosan, we co-crystallized LGK
The similar arrangement of levoglucosan in the active site
with ADP and magnesium and then soaked the grown crystals A-chain when compared with the closed AnmK structure sug-
with 100 mM levoglucosan. Despite the fact that levoglucosan is gests that the sugar cleavage mechanism is conserved between
smaller than anhMurNAc because it has hydroxyls in place of LGK and AnmK. For AnmK, Asp-182 acts as a base to enhance
the bulkier lactyl and acetamido moieties of anhMurNAc, the the nucleophilicity of a water molecule (WatA), which then
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Structural Insights into Levoglucosan Bioconversion
B
A
Tyr234
Tyr234
Aspr237
Asp26
Asp26
M2
ADP
ADP
Asp221
Asp221
Gly328
Gly328
Glu362
Glu362
Gly189
Asn192
Gly189
LG
LG
alternate
orientation
alternate
orientation
Asn192
WatA
WatA
Asp212
Asp212
D
C
Asn192
LG
LG
alternate
orientation
WatA
WatA
Asp212
Asp212
Thr116
Thr116
FIGURE 4. A–C, LGK active site structures with levoglucosan bound in an alternate orientation. D, additional crystal form with levoglucosan bound in a
catalytically conducive orientation. mFo Ϫ DFc omit maps are shown at 2.5 (A and B) or 3.0␴ (C and D).
attacks the anomeric carbon of the sugar resulting in cleavage of demonstrated an accumulation of the ␣ anomer of anhMur-
the 1,6-anhydro bond (24). WatA also appears ideally posi- NAc following catalysis using similar techniques (24). It is
tioned to attack the anomeric carbon in the LGK structure (Fig. apparent that levoglucosan is less stable during the catalytic
3, A and C). Consistent with this observation, mutation of the reaction, because it is held in the active site through fewer
predicted catalytic base in LGK, Asp-212, to an asparagine bonding interactions when compared with anhMurNAc bound
results in complete abrogation of LGK activity as evidenced by to AnmK. We propose that in addition to cleavage of the 1,6-
ϩ
an NADP -coupled assay. As another means to follow the pro- anhydro ring, the pyranose ring of the levoglucosan is opened
1
gress of the LGK reaction, H NMR experiments were per- when the anomeric carbon of levoglucosan migrates from its
1
4
formed using a coupled system whereby ATP is regenerated position in a C4 to a C1 conformation. It is plausible that
from the LGK reaction ADP product using pyruvate kinase as increased conformational strain during this reaction results in
the phosphotransfer enzyme and phosphoenolpyruvic acid as the linear form of G6P as the initial product, which then equil-
the phosphoryl donor. Importantly, this method allows for the ibrates to a mixture of both anomeric forms (Fig. 3E).
direct observation of levoglucosan consumption and G6P pro-
LGK Binds Levoglucosan in Two Distinct Orientations—In
duction during the reaction. The reaction showed fast initial contrast to the A-chain monomeric structure, the levoglucosan
build-up of G6P (within 2 min) followed by a slower linear sugar substrate was also found to bind to LGK in an alternate
phase until the phosphoenolpyruvic acid was depleted (Fig. 3, D orientation in the B-chain monomeric subunit, whereby it is
and E). Both ␣ and ␤ anomers of G6P appear simultaneously in rotated by ϳ135o with regard to the plane of the pyranose sugar
the reaction mixture and at a ratio corresponding to the equi- (Figs. 3A and 4A). Both conformations form hydrogen bonds
librium ratio of anomers for G6P. The turnover rate of the LGK with Asp-212, whereas the alternate conformation in the
reaction calculated using this technique is 0.14/s at 20 °C, which B-chain is supported by an additional hydrogen bond between
is faster than the reported anomerization rates of G6P of 0.04/s Asn-192 and the O3 oxygen of levoglucosan. Interestingly, the
at 20 °C (42). This suggests that the initial product of the LGK magnesium ions are not bound in this structure and the B-fac-
reaction is G6P in its linear form, because we would expect an tors (Table 1) and corresponding electron density for ADP are
accumulation of the ␣ or ␤ anomer following catalysis if either much poorer than in the A-chain structure. In the lower affinity
anomer were formed during the reaction. These results are in ADP binding site, when compared with the other structures,
contrast to those obtained using AnmK as the enzyme, which several bonds either are abrogated or show longer bonding dis-
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Structural Insights into Levoglucosan Bioconversion
tances. Amino acids Asp-26 and Glu-362 are no longer bound standing the cellular processes they regulate or control. The
to magnesium ions, and these residues instead form bonds crystallographic structures of LGK described here provide
directly with the phosphates of ADP (Fig. 4A). The two domains insight into the unique reaction mechanism carried out by
of the B-chain in this structure also appear slightly more open anhydrosugar kinases that involves both cleavage and
than the A-chain, with a 3° rotation of the two domains calcu- phosphorylation of their sugar substrate. A somewhat unex-
lated using the program DynDom (43), suggesting a possible pected observation for LGK was the binding of two magnesium
reciprocal effect between ligand binding and protein domain ions in the enzyme active site. The binding of two magnesiums
movements.
has been observed in other kinases and is common in protein
Our initial efforts to determine the structure of LGK with kinases (i.e. CDK2 kinase (44, 45)). However, to our knowledge,
bound levoglucosan thus demonstrated two binding modes for this is the first observation of two magnesium binding sites in
the sugar. Because this was an intriguing result, we sought to the hexokinase family. The M2 site appears to assist in stabiliz-
determine additional crystal forms of LGK with levoglucosan ing the positions and geometrical alignment of the nucleotide
bound to determine whether these results could be duplicated phosphate groups, because it forms bonding interactions with
in another crystal form. After extensive crystallization screen- both the ␣- and ␤-phosphates of the ADP, although a role in
ing, we found another crystallization condition for LGK in the charge stabilization or modulation during the catalytic reaction
presence of ADP and magnesium that uses either ammonium is also plausible. It is probable that the presence of the M1 site is
sulfate or sodium malonate as the precipitant, instead of PEG also required for maintenance of charges that develop during
4000 that was used for the original structures. We pursued this the reaction by enhancing the electrophilicity of the departing
crystal form with the intention to determine additional struc- ␥-phosphate or by acting as a transient bridge between the
tures of LGK bound to levoglucosan in either of the previous ␤-phosphate of ADP and the phosphoryl group of the product
observed orientations. LGK in this form crystallizes in the same during the reaction as suggested for other kinases (46). The
P41212 space group as the originally determined structures, binding of magnesium in other hexokinases such as Sulfolobus
although with different unit cell dimensions (Table 1), and with tokodaii hexokinase is also roughly equivalent in structural
its dimeric interface lying on a crystal symmetry axis. The struc- rearrangement to the M1 site, with magnesium binding to a
ture in this case was determined to 2.0 Å as a monomer in the ␤-phosphate oxygen of ADP, suggesting a shared functional
asymmetric unit, with both monomers of the physical dimer evolution (47). For other kinases that use two magnesiums for
essentially being determined as identical structures due to the catalysis such as the protein kinase CDK2, it has been shown
crystal symmetry. Our initial attempts to solve the structure that the resulting product ADP is slowly released from the
with ADP, aluminum fluoride, and magnesium present during active site following catalysis (45). The need by LGK to utilize
co-crystallization followed by soaking with 200 mM levogluco- two magnesium ions for catalysis may also come at a cost of
san resulted in a structure similar to that observed in the lowering the rate of product release, although further more
B-chain of the dimeric structure, with levoglucosan bound in extensive kinetic studies will be required to evaluate the rate. In
the alternate orientation, ADP showing poor electron density, CDK2, it was shown that magnesium binds less tightly to the
and no visible bound aluminum fluoride (Fig. 4B). Only the M2 M1 site, which is consistent with the observation that in LGK,
magnesium appears bound in this structure, although with only the M2 magnesium is bound in the monomeric structure
higher B-factors than in the other Mg-containing structures bound to ADP and levoglucosan (45). Although the compara-
(Table 1).
tive binding affinities of the two magnesiums remain unclear, it
Although levoglucosan in the alternate orientation makes is likely that higher concentrations of magnesium would
two direct hydrogen bonds to LGK (Asp-212 and Asn-192), a increase the relative occupancy of both magnesium sites in
similar water-bonding arrangement with Thr-116 to that LGK and thereby limit ADP product release.
observed in the catalytically conducive orientation appears to
In our previous work, the position of magnesium could not
be maintained. However, in this orientation, two water mole- unambiguously be resolved in the closed AnmK structure
cules are required to bridge the gap to the levoglucosan ring bound to ADP (24). However, a single magnesium appeared to
oxygen, which may only act as a hydrogen bond acceptor (Fig. be bound in one of the open AnmK structures bound to
4C). We were eventually also able to solve another structure of AMPPCP (25). It is plausible that AnmK also uses two magne-
LGK, to 1.85 Å, with levoglucosan bound in the catalytically siums during catalysis, because the nucleotide binding site
conducive conformation by co-crystallizing the enzyme with structures are highly conserved between the two enzymes and
200 mM levoglucosan in the monomeric crystal form without the one magnesium observed in AnmK was coordinated by the
the nucleotide or magnesium present (Fig. 4D). These addi- same glutamate residue (Glu-326 and Glu-362 in AnmK and
tional structures support the initial observations of two distinct LGK, respectively) that is key to the M1 binding. The inability
binding modes of levoglucosan to LGK.
to view both magnesiums in the closed AnmK structures may
be due to several factors including the lower resolution of the
AnmK structure (2.23 Å) or the relatively high B-factors for
Discussion
Enzyme-driven phosphoryl transfer is a fundamental bio- ADP bound to AnmK (41.3 and 56.9 for either monomer). The
chemical reaction that is carried out through a surprisingly protein and water interactions that govern magnesium binding
variable array of mechanisms, the intricacies of which are often may also be influenced by the pH of the crystallization condi-
only made apparent when examined at the structural level. A tions, which was 5.6 for the closed AnmK structure and 7.5 for
detailed understanding of these reactions is crucial to under- the LGK structure bound to two magnesiums, the latter of
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Structural Insights into Levoglucosan Bioconversion
which represents a form of the structure at physiological pH. extent, a possible rationale for the alternate orientation of levo-
Taken together our observations suggest that ADP is bound to glucosan may be to abrogate water-bonding networks in the
the AnmK structure in a lower affinity state, similar to the LGK active site that are also required for the octahedral coordination
structures in which magnesium ions were not observed.
with magnesium, displacing the metals and also promoting the
Additional unusual observations for the LGK structures are release of ADP. With the knowledge that ADP is a product
the two binding orientations for the levoglucosan substrate, inhibitor of LGK (KI ϭ 0.2 mM (51)) and G6P is not (13, 51), it is
which were both observed in two different crystal forms, and likely that G6P is quickly released following catalysis. This sug-
the relatively few direct hydrogen bonds the enzyme makes gests the possibility that the active site can be reoccupied by
with the substrate. Although the related enzyme AnmK binds another molecule of levoglucosan prior to ADP and magnesium
its sugar substrate with a total of five hydrogen bonds (24), LGK release. A more detailed analysis of water orientations in the
mediates only one or two hydrogen bonds (catalytically condu- enzyme structure, including visualization of hydrogen atoms
cive or the alternate arrangement, respectively) with its sugar and their associated molecular interactions, using methods
substrate. These observations are also supported by the mark- such as neutron crystallography would allow for a more thor-
edly differing Km values of the respective sugar substrates of ough investigation into these mechanistic details (52).
AnmK and LGK enzymes. However, this does not explain why
Summary and Conclusions
LGK has not evolved residues in the active site that would
In addition to a detailed examination of the structure and
mechanism of anhydrosugar kinases that elaborates on the
function of these enzymes in natural biological processes, these
studies provide a blueprint for future efforts to engineer LGK
enzymes as catalysts in practical applications, such as biofuel
production. Mutations that alter the substrate or product affin-
ity of the enzyme could lead to a greater catalytic efficiency for
the bioconversion of levoglucosan. However, possible rational
approaches may be hampered by the dual binding orientations
of levoglucosan, and natural selection has not yet provided a
better solution to the high intrinsic Km of LGK for its sugar
substrate, suggesting that such efforts to improve affinity for
the sugar may be very challenging. Combining structural data
with advanced protein engineering and computational design
may however yet hold promise for improving LGK to produce
levoglucosan-derived biofuel.
improve its affinity for levoglucosan by imposing more bonding
interactions with the substrate. Although we cannot rule out
that the unusually high Km for levoglucosan is not rate-limiting
to the catalytic efficiency of the enzyme, we propose that the
comparatively high Km for LGK is intrinsically linked to the
ability of the enzyme to alternate the levoglucosan binding ori-
entations. This structural requirement may prohibit selective
mutations of residues in the active site that would support addi-
tional direct bonds between LGK and the sugar, which might
enhance binding affinity, but would limit its ability to bind the
sugar in two orientations. This property may also make the
enzyme more prone to competitive inhibition, as evidenced by
the binding of Tris in the active site and our kinetic analyses
(Fig. 2, E and F). Other enzymes that have been shown to bind
their substrate in multiple orientations do so with varying affin-
ity and functionally are quite diverse, making it difficult to com-
pare the mechanism of substrate binding for these enzymes
(48–50).
Author Contributions—J. P. B., T. A. W., B. L. M., C. J. U., and R. M.
designed the research; J. P. B., J. R. K., and R. M. performed the
research; J. P. B., L. R. J., and R. M. wrote the manuscript. All authors
contributed to analysis of the results and editing of the manuscript.
The final version of the manuscript was approved by all of the
authors.
The high Km of LGK for levoglucosan may also at least par-
tially be due to the abundance of levoglucosan in the natural
environment, particularly in areas that have been subjected to
biomass burning events. In this context, the possibility of weak
selection pressure to improve catalytic efficiency could also be
influenced by the use of levoglucosan as only a secondary metab-
olite that is not absolutely required for growth or survival. LGK
may also be somewhat promiscuous toward or have significant
catalytic activity for other anhydrosugars. Although AnmK has
been shown not to be able to use levoglucosan as a substrate,
recombinantgrowthstudiesusingE. colihaveshownthatLGKhas
Acknowledgments—We thank Shaun Labiuk, Pavel Afonine, and the
Canadian Light Source as well as the staff at the SLAC National
Accelerator Laboratory for assistance in data collection and pro-
cessing. We thank the Los Alamos National Laboratory Principal
Associate Directorate for Science, Technology and Engineering for
institutional investment in key equipment that enabled NMR mea-
weak activity for anhGlcN (1,6-anhydro-␤-D-glucosamine) and surements reported here. The Canadian Light Source is supported by
Natural Sciences and Engineering Research Council of Canada
(NSERC), the National Research Council, Canadian Institutes of
Health Research, and the University of Saskatchewan. Use of the Stan-
ford Synchrotron Radiation Lightsource, SLAC National Accelerator
Laboratory, is supported by the U. S. Department of Energy (DOE), Office
of Science, Office of Basic Energy Sciences under Contract DE-AC02-
76SF00515. The Stanford Synchrotron Radiation Lightsource Structural
Molecular Biology Program is supported by the DOE Office of Biological
and Environmental Research, and by the National Institutes of Health,
NIGMS (including Grant P41GM103393). We also thank Brian Broom-
Peltz for technical assistance and Dr. Virginia A. Unkefer for help with
editing the manuscript.
anhGlcNAc 1,6-anhydro-N-acetyl-␤-D-glucosamine (27). How-
ever, it does not appear that these sugars can be used as a carbohy-
drate source by L. starkeyi, suggesting that they are not trans-
ported into the cell or that metabolic pathways do not exist in
Lipomyces to further utilize the resulting products (27).
In LGK, the making and breaking of water-bonding arrange-
ments and dipole interactions not only play an intrinsic role in
coordinating the octahedral networks required for magnesium
coordination, but they also play a key role in mediating interac-
tions with levoglucosan. This is apparent from the water-bond-
ing network between levoglucosan and Thr-116 that appears to
be maintained in both levoglucosan orientations. To this
26646 JOURNAL OF BIOLOGICAL CHEMISTRY
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Structural Insights into Levoglucosan Bioconversion
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26648 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 290•NUMBER 44•OCTOBER 30, 2015

Producing Glucose 6-Phosphate from Cellulosic Biomass: STRUCTURAL
INSIGHTS INTO LEVOGLUCOSAN BIOCONVERSION
John-Paul Bacik, Justin R. Klesmith, Timothy A. Whitehead, Laura R. Jarboe, Clifford
J. Unkefer, Brian L. Mark and Ryszard Michalczyk
J. Biol. Chem. 2015, 290:26638-26648.
doi: 10.1074/jbc.M115.674614 originally published online September 9, 2015
Access the most updated version of this article at doi: 10.1074/jbc.M115.674614
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