Structure, Vol. 12, 1619–1629, September, 2004, 2004 Elsevier Ltd. All rights reserved.DOI 10.1016/j.str.2004.06.020
Novel Catalytic Mechanism of Glycoside Hydrolysis
Based on the Structure of an NAD?/Mn2?-Dependent
Phospho-?-Glucosidase from Bacillus subtilis
(M6P) is hydrolyzed by GlvA, yielding glucose and glu-
cose 6-phosphate (G6P), both of which can enter the
energy-generating glycolytic pathway (Thompson et al.,
GlvA is a 6-phospho-?-glucosidase belonging to fam-
ily 4 of the glycoside hydrolases (GH4 [Coutinho and
Henrissat, 1999]). GH4 enzymes are unique in their re-
quirement for NAD(H) and a divalent metal ion for activ-
ity, unlike glycoside hydrolases from other families
(Thompson et al., 1998). The GH4 family is also unique
in that it includes both ?-glycosidases, such as GlvA
(Lapidus et al., 1997) from B. subtilis, and ?-glycosi-
dases, such as the 6-phospho-?-glucosidase CelF
(Thompson et al., 1999) from Escherichia coli (Figure 1;
Supplemental Figure SA). Members of this family can
hydrolyze both phosphorylated (GlvA) and nonphos-
phorylated (galactosidase) disaccharides, as is the case
with family 1 glycosidases (Coutinho and Henrissat,
1999), as well as a variety of other natural and synthetic
?- and ?-glucosides (Thompson et al., 1995, 1998, 1999,
2001a, 2001b). The coenzyme NAD(H) does not appear
to be altered following the reaction (Thompson et al.,
1998), and its role in catalysis has thus far been unclear.
The unique appearance of both ?- and ?-glycosidases
within one family and the requirement for NAD(H) sug-
gest a novel catalytic mechanism for this group of gly-
cosidases (Thompson et al., 1995, 1998, 1999, 2001a,
2001b; Varrot et al., 1999; Bouma et al., 1997).
There are currently two known glycosidase mecha-
nisms, resulting in either net inversion or retention of
anomeric configuration as suggested by Koshland 50
out a direct displacement using a pair of carboxylic
acids separated by 6–12 A˚as acid and base catalysts.
Retaining glycosidases use a double-displacement
mechanismin whichthe 5A˚separationof thecarboxylic
acids permits an initial acid-catalyzed formation of a
covalent glycosyl-enzyme intermediate that subse-
quently undergoes base-catalyzed hydrolysis (Zechel
and Withers, 2000), both steps occurring via oxocar-
benium ion-like transition states.
We have determined the crystal structure of GlvA in
complex with NAD(H), Mn2?, and the reaction product
G6P to a resolution of 2.05A˚. The structureof GlvA shows
no similarity to that of any known glycosidases outside
of the GH4 family, but does show resemblance to those
of lactate/malate dehydrogenases, despite low (?15%)
sequence identity. Another ?-glucosidase of the GH4
family, AglA from Thermotoga maritima, first showed
nases extends beyond the NAD(H) binding (Rossmann
the GH4 enzymes and the organic acid dehydrogenases
diverged from a common precursor that subsequently
adapted mechanistically to different metabolic require-
The similarity to the dehydrogenases, particularly the
Shyamala S. Rajan,1Xiaojing Yang,1Frank Collart,2
Vivian L.Y. Yip,3Stephen G. Withers,3
Annabelle Varrot,4John Thompson,5
Gideon J. Davies,4and Wayne F. Anderson1,*
1Molecular Pharmacology and Biological Chemistry
Feinberg School of Medicine
Chicago, Illinois 60611
Argonne National Laboratory
Argonne, Illinois 60439
3Department of Chemistry
University of British Columbia
Vancouver, BC V6T 1Z1
4Department of Chemistry
University of York
Heslington, York Y015DD
5Microbial Biochemistry and Genetics Unit
Oral Infection and Immunity Branch
National Institutes of Health
Bethesda, Maryland 20892
GlvA, a 6-phospho-?-glucosidase from Bacillus subti-
lis, catalyzes the hydrolysis of maltose-6?-phosphate
and belongs to glycoside hydrolase family GH4. GH4
enzymes are unique in their requirement for NAD(H)
and a divalent metal for activity. We have determined
to 2.05 A˚resolution. Analyses of the active site archi-
tecture, in conjunction with mechanistic studies and
precedent from the nucleotide diphosphate hexose
dehydratases and other systems, suggest a novel
mechanism of glycoside hydrolysis by GlvA that in-
volves both the NAD(H) and the metal.
Carbohydrate assimilation in both Gram-negative and
Gram-positive bacteria is frequently initiated via the
phosphoenolpyruvate-dependent sugar phosphotrans-
ferasesystem (PEP-PTS).ThePEP-PTSconsists ofsev-
eral cytoplasmic and membrane proteins that together
enable the simultaneous phosphorylation and transport
of sugars across the cytoplasmic membrane (Meadow
et al.,1990). This systemis the primary routefor maltose
translocation in B. subtilis and is regulated by the glv
operon (Yamamoto et al., 2001). Maltose is transported
into the cytoplasm, concomitant with its phosphoryla-
tion by GlvC, a membrane-anchored permease (Yama-
moto et al., 2001). Intracellular maltose-6?-phosphate
Figure 1. Sequence Alignment of GH4 Family and Comparison with LDH
Representative sequences from subsets (?-glucosidases, galactosidases, and ?-glucosidases) of GH4 enzymes are aligned: AgaL-Bs, an
?-galactosidase from B. subtilis; CelF-Ec, a ?-glucosidase from E. coli. Structure-based sequence alignment of LDH (from P. falciparum;
1LDG) and AglA (a GH4 enzyme from T. maritima; 1OBB) with GlvA (from B. subtilis; GlvA-Bs) is also shown. Red asterisks mark residues
involved in catalysis and in the coordination of the metal ion. Secondary structural elements shown at the top of the sequences are those of
GlvA. The 1? and 2? structures of GlvA are color-coded as follows: N-terminal Rossmann fold region A in blue and the following region B in
of GlvA with AglA and LDH were done using algorithms implemented in PdbViewer (Guex and Peitsch, 1997).
retentionof thesameactive-sitearchitecture, andalack
of similarity to glycoside hydrolases of other families
provided another indication that GH4 enzymes employ
a novel mechanism of catalysis. On the basis of struc-
tural and mechanistic studies, we propose a reaction
stabilization of intermediates by Mn2?. The proposed
mechanism also satisfies the varied substrate stereo-
chemistry seen in reactions catalyzed by GH4 enzymes.
Part of the proposed mechanism bears some similarity
Family 4 Glycosidase Structure and Mechanism
Corpet, F. (1998). Multiple sequence alignment with hierarchical
clustering. Nucleic Acids Res. 16, 10881–10890.
Coutinho, P.M., and Henrissat, B. (1999). Carbohydrate-active en-
zymes: an integrated database approach. In Recent Advances in
Carbohydrate Engineering, H.J. Gilbert, G.J. Davies, B. Svensson,
and B. Henrissat, eds. (Cambridge, UK: Royal Society of Chemistry),
Dieckman, L., Gu, M., Stols, L., Donnelly, M.I., and Collart, F.R.
(2002). High throughput methods for gene cloning and expression.
Protein Expr. Purif. 25, 1–7.
Doublie, S. (1997). Preparation of selenomethionyl proteins for
phase determination. Methods Enzymol. 276, 523–530.
Dunn, C.R., Banfield, M.J., Barker, J.J., Higham, C.W., Moreton,
K.M., Turgut-Balik, D., Brady, R.L., and Holbrook, J.J. (1996). The
structure of lactate dehydrogenase from Plasmodium falciparum
reveals a new target for anti-malarial design. Nat. Struct. Biol. 3,
Esnouf, R.M. (1997). An extensively modified version of MolScript
that includes generally enhanced coloring capabilities. J. Mol.
Graph. Model. 15, 132–134.
Gouet, P., Courcelle, E., Stuart, D.I., and Metoz, F. (1999). ESPript:
analysis of multiple sequence alignments in PostScript. Bioinforma-
tics 15, 305–308.
Guex, N., and Peitsch, M.C. (1997). SWISS-MODEL and the Swiss-
PdbViewer: an environment for comparative protein modeling. Elec-
trophoresis 18, 2714–2723.
Hall, M.D., and Banaszak, L.J. (1993). Crystal structure of a ternary
complex of Escherichia coli malate dehydrogenase citrate and NAD
at 1.9 A˚resolution. J. Mol. Biol. 232, 213–222.
Harding, M.M. (2001). Geometry of metal-ligand interactions in pro-
teins. Acta Crystallogr. D Biol. Crystallogr. 57, 401–411.
Holm, L., and Sander, C. (1993). Protein structure comparison by
alignment of distance matrices. J. Mol. Biol. 233, 123–138.
Koshland, D.E. (1953). Stereochemistry and the mechanism of enzy-
matic reactions. Biol. Rev. Cam. Phil. Soc. 28, 416–436.
Kraulis, P.J. (1991). MOLSCRIPT—a program to produce both de-
tailed and schematicplots of protein structures.J. Appl. Crystallogr.
Lapidus, A., Galleron, N., Sorokin, A., and Ehrlich, S.D. (1997). Se-
quencing and functional annotation of the Bacillus subtilis genes in
the 200 kb rrnB-dnaB region. Microbiol. 143, 3431–3441.
Lee, S.S., Yu, S., and Withers, S.G. (2002). ?-1,4-Glucan lyase per-
forms a trans-elimination via a nucleophilic displacement followed
by a syn-elimination. J. Am. Chem. Soc. 124, 4948–4949.
Lodge, J.A., Maier, T., Liebl, W., Hoffmann, V., and Stra ¨ter, N. (2003).
Crystal structure of Thermotoga maritima ?-glucosidase AglA de-
fines a new clan of NAD?-dependent glycosidases. J. Biol. Chem.
Lunin, V.V., Li, Y., Linhardt, R.J., Miyazono, H., Kyogashima, M.,
Kaneko, T., Bell, A.W., and Cygler, M. (2004). High-resolution crystal
structure of Arthrobacter aurescens chondroitin AC lyase: an en-
zyme-substrate complex defines the catalytic mechanism. J. Mol.
Biol. 337, 367–386.
McRee, D.E. (1999). XtalView/Xfit—a versatile program for manipu-
lating atomic coordinates and electron density. J. Struct. Biol. 125,
Meadow, N.D., Fox, D.K., and Roseman, S. (1990). The bacterial
phosphoenol-pyruvate: glycose phosphotransferase system. Annu.
Rev. Biochem. 59, 497–542.
ular graphics. Methods Enzymol. 277, 505–524.
Murshudov, G.N., Vagin, A.A., and Dodson, E.J. (1997). Refinement
of macromolecular structures by the maximum-likelihood method.
Acta Crystallogr. D Biol. Crystallogr. 53, 240–255.
Otwinowski,Z., andMinor, W.(1997). Processingof X-raydiffraction
data collected in oscillation mode. Methods Enzymol. 276, 307–326.
Raasch, C., Armbrecht, M., Streit, W., Ho ¨cker, B., Stra ¨ter, N., and
Liebl, W. (2002). Identification of residues important for NAD?bind-
ing by the Thermotoga maritima ?-glucosidase AglA, a member of
glycoside hydrolase family 4. FEBS Lett. 517, 267–271.
M.I. (2002). A new vector for high-throughput, ligation-independent
cloning encoding a tobacco etch virus protease cleavage site. Pro-
tein Expr. Purif. 25, 8–15.
Terwilliger,T.C.,and Berendzen,J.(1999).Automated MADandMIR
structure solution. Acta Crystallogr. D Biol. Crystallogr. 55, 849–861.
Thoden, J.B., Wohlers, T.M., Fridovich-Keil, J.L., and Holden, H.M.
(2000). Crystallographic evidence for Tyr 157 functioning as the
active site base in human UDP-galactose 4-epimerase. Biochemis-
try 39, 5691–5701.
Thompson, J., Gentry-Weeks, C.R., Nguyen, N.Y., Folk, J.E., and
Robrish, S.A. (1995). Purification from Fusobacterium mortiferum
ATCC-25557 of a 6-phosphoryl-O-?-D-glucopyranosyl:6-phospho-
glucohydrolase that hydrolyzes maltose-6?-phosphate and related
phospho-?-D-glucosides. J. Bacteriol. 177, 2505–2512.
Thompson, J., Pikis, A., Ruvinov, S.B., Henrissat, B., Yamamoto, H.,
and Sekiguchi, J. (1998). The gene glvA of Bacillus subtilis 168
encodes a metal-requiring, NAD(H)-dependent 6-phospho-?-glu-
cosidase—assignment to Family 4 of the glycosylhydrolase super-
family. J. Biol. Chem. 273, 27347–27356.
Thompson, J., Ruvinov, S.B., Freedberg, D.I., and Hall, B.G. (1999).
Cellobiose-6-phosphate hydrolase (CelF) of Escherichia coli: char-
acterization and assignment to the unusual Family 4 of glycosylhy-
drolases. J. Bacteriol. 181, 7339–7345.
Thompson, J., Robrish, S.A., Immel, S., Lichtenthaler, F.W., Hall,
B.G., and Pikis, A. (2001a). Metabolism of sucrose and its five link-
participation and properties of sucrose-6-phosphate hydrolase and
phospho-?-glucosidase. J. Biol. Chem. 276, 37415–37425.
Thompson, J., Robrish, S.A., Pikis, A., Brust, A., and Lichtenthaler,
F.W. (2001b). Phosphorylation and metabolism of sucrose and its
five linkage-isomeric ?-D-glucosyl-D-fructoses by Klebsiella pneu-
moniae. Carbohydr. Res. 331, 149–161.
Varrot, A., Yamamoto, H., Sekiguchi, J., Thompson, J., and Davies,
G.J. (1999). Crystallization and preliminary X-ray analysis of the
6-phospho-?-glucosidase from Bacillus subtilis. Acta Crystallogr. D
Biol. Crystallogr. 55, 1212–1214.
Wilson, G., and Fox, C.F. (1974). The ?-glucoside system of Esche-
richia coli. IV. Purification and properties of phospho-?-glucosi-
dases A and B. J. Biol. Chem. 249, 5586–5598.
Regulation of the glv operon in Bacillus subtilis: YfiA (GlvR) is a
positive regulator of the operon that is repressed through CcpA and
cre. J. Bacteriol. 183, 5110–5121.
Yip, V.L.Y., Varrot, A., Davies, G.J., Rajan, S.S., Yang, X., Thompson,
J., Anderson, W.F., and Withers, S.G. (2004). An unusual mechanism
of glycoside hydrolysis involving redox and elimination steps by a
family 4 ?-glycosidase from Thermotoga maritima. J. Am. Chem.
Soc. 126, 8354–8355.
Zechel, D.L., and Withers, S.G. (2000). Glycosidase mechanisms:
anatomy of a finely tuned catalyst. Acc. Chem. Res. 33, 11–18.
Data Bank under the accession code 1U8X.