DB code: D00827

RLCP classification 9.5010.194230.8500 : Hydride transfer
8.113.904850.8015 : Isomerization
4.1010.695600.8301 : Addition
5.1020.2074300.8301 : Elimination
8.131.704320.8015 : Isomerization
9.1050.192630.8500 : Hydride transfer
CATH domain 3.40.50.720 : Rossmann fold
3.90.110.10 : L-2-Hydroxyisocaproate Dehydrogenase; Chain A, domain 2 Catalytic domain
E.C. 3.2.1.122
CSA
M-CSA
MACiE

CATH domain Related DB codes (homologues)
3.90.110.10 : L-2-Hydroxyisocaproate Dehydrogenase; Chain A, domain 2 D00005 D00008 M00171
3.40.50.720 : Rossmann fold S00543 S00551 S00552 S00553 S00602 S00604 S00605 S00608 S00610 S00625 S00319 S00328 S00329 S00330 S00331 S00332 D00456 D00457 D00458 S00324 S00320 S00325 S00326 S00327 D00459 S00335 S00336 S00334 T00219 S00339 D00513 D00001 D00002 D00003 D00005 D00007 D00008 D00010 D00012 D00017 D00018 D00023 D00027 D00028 D00031 D00032 D00033 D00034 D00035 D00037 D00048 D00071 D00476 D00481 D00482 D00490 D00492 D00494 D00545 D00601 D00603 D00604 D00605 D00615 D00845 D00857 D00858 M00161 M00171 M00210 T00002 T00010 T00011 T00015 T00227 T00247 T00408 T00414 D00262 D00274 D00275 M00035 T00109

Uniprot Enzyme Name
UniprotKB Protein name Synonyms RefSeq CAZy Pfam
P54716 Maltose-6''-phosphate glucosidase
EC 3.2.1.122
6-phospho-alpha-D-glucosidase
6-phosphoryl-O-alpha-D-glucopyranosyl:phosphoglucohydrolase
NP_388699.1 (Protein)
NC_000964.3 (DNA/RNA sequence)
GH4 (Glycoside Hydrolase Family 4)
PF02056 (Glyco_hydro_4)
PF11975 (Glyco_hydro_4C)
[Graphical View]

KEGG enzyme name
Maltose-6'-phosphate glucosidase
Phospho-alpha-glucosidase

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P54716 GLVA_BACSU Maltose 6'-phosphate + H(2)O = D-glucose + D-glucose 6-phosphate. Homotetramer. Binds 1 divalent metal ion such as manganese, iron, cobalt or nickel per subunit. Binds 1 NAD per subunit.

KEGG Pathways
Map code Pathways E.C.
MAP00500 Starch and sucrose metabolism

Compound table
Cofactors Substrates Products Intermediates
KEGG-id C00034 C00003 C02995 C00001 C00031 C00668 I00113 I00114 I00115 I00116 I00117
E.C.
Compound Manganese NAD+ maltose 6'-phosphate H2O D-glucose alpha-D-Glucose 6-phosphate Maltose 3'-dehydro-6'-phosphate 2,3-ene-6-phosphate-alpha-D-allopyranosyl-(1->4)-D-glucose D-glucose-1-deoxy-3-dehydro-6-phosphate-1,2-ene alpha-D-allose-2,3-ene-6-phosphate alpha-D-glucose-3-dehydro-6-phosphate
Type heavy metal amide group,amine group,nucleotide phosphate group/phosphate ion,polysaccharide H2O carbohydrate carbohydrate,phosphate group/phosphate ion
ChEBI 18291
35154
15846
15703
15377
4167
17665
PubChem 23930
5893
439874
22247451
962
5793
439284
1nrhX01 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:NAD Unbound Unbound Unbound Unbound Unbound Unbound Unbound Unbound
1u8xX01 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Unbound Bound:NAD Unbound Unbound Unbound Unbound Unbound Unbound Unbound Unbound
1nrhX02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MN Unbound Unbound Unbound Analogue:G6P Unbound Unbound Unbound Unbound Unbound
1u8xX02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain Bound:_MN Unbound Unbound Unbound Analogue:G6P Unbound Unbound Unbound Unbound Unbound

Reference for Active-site residues
resource references E.C.
Literature [3], [9] & Swiss-prot;P54716

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1nrhX01 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 111
1u8xX01 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain GLU 111
1nrhX02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain ASP 172;TYR 265 CYS 171;HIS 202 (Manganese binding)
1u8xX02 Pdbj logo s Rasmollogo id Rasmollogo chain Mmcif id Mmcif chain ASP 172;TYR 265 CYS 171;HIS 202 (Manganese binding)

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[3]
Figure 4, Figure 5, p.1626-1627
[8]
Figure 1, p.147-149
[9]
Figure 1, p.9851
[10]
Figure 7, p.535-537

References
[1]
Resource
Comments
Medline ID
PubMed ID 9765262
Journal J Biol Chem
Year 1998
Volume 273
Pages 27347-56
Authors Thompson J, Pikis A, Ruvinov SB, Henrissat B, Yamamoto H, Sekiguchi J
Title The gene glvA of Bacillus subtilis 168 encodes a metal-requiring, NAD(H)-dependent 6-phospho-alpha-glucosidase. Assignment to family 4 of the glycosylhydrolase superfamily.
Related PDB
Related UniProtKB
[2]
Resource
Comments
Medline ID
PubMed ID 15237973
Journal J Am Chem Soc
Year 2004
Volume 126
Pages 8354-5
Authors Yip VL, Varrot A, Davies GJ, Rajan SS, Yang X, Thompson J, Anderson WF, Withers SG
Title An unusual mechanism of glycoside hydrolysis involving redox and elimination steps by a family 4 beta-glycosidase from Thermotoga maritima.
Related PDB
Related UniProtKB
[3]
Resource
Comments X-RAY CRYSTALLOGRAPHY (2.05 ANGSTROMS) OF COMPLEX WITH NAD(H) AND ALPHA-D-GLUCOSE-6-PHOSPHATE, REACTION MECHANISM.
Medline ID
PubMed ID 15341727
Journal Structure
Year 2004
Volume 12
Pages 1619-29
Authors Rajan SS, Yang X, Collart F, Yip VL, Withers SG, Varrot A, Thompson J, Davies GJ, Anderson WF
Title Novel catalytic mechanism of glycoside hydrolysis based on the structure of an NAD+/Mn2+ -dependent phospho-alpha-glucosidase from Bacillus subtilis.
Related PDB 1u8x
Related UniProtKB P54716
[4]
Resource
Comments
Medline ID
PubMed ID 16263268
Journal Curr Opin Struct Biol
Year 2005
Volume 15
Pages 637-45
Authors Davies GJ, Gloster TM, Henrissat B
Title Recent structural insights into the expanding world of carbohydrate-active enzymes.
Related PDB
Related UniProtKB
[5]
Resource
Comments
Medline ID
PubMed ID 15670594
Journal J Mol Biol
Year 2005
Volume 346
Pages 423-35
Authors Varrot A, Yip VL, Li Y, Rajan SS, Yang X, Anderson WF, Thompson J, Withers SG, Davies GJ
Title NAD+ and metal-ion dependent hydrolysis by family 4 glycosidases: structural insight into specificity for phospho-beta-D-glucosides.
Related PDB
Related UniProtKB
[6]
Resource
Comments
Medline ID
PubMed ID
Journal Biocatalysis and Biotransformation
Year 2006
Volume 24
Pages 167-176
Authors Yip VLY, Withers SG
Title Family 4 glycoside hydrolases are special: The first beta-elimination mechanism amongst glycoside hydrolases
Related PDB
Related UniProtKB
[7]
Resource
Comments
Medline ID
PubMed ID 16401086
Journal Biochemistry
Year 2006
Volume 45
Pages 571-80
Authors Yip VL, Withers SG
Title Mechanistic analysis of the unusual redox-elimination sequence employed by Thermotoga maritima BglT: a 6-phospho-beta-glucosidase from glycoside hydrolase family 4.
Related PDB
Related UniProtKB
[8]
Resource
Comments
Medline ID
PubMed ID 16495121
Journal Curr Opin Chem Biol
Year 2006
Volume 10
Pages 147-55
Authors Yip VL, Withers SG
Title Breakdown of oligosaccharides by the process of elimination.
Related PDB
Related UniProtKB
[9]
Resource
Comments REACTION MECHANISM, CHARACTERIZATION, COFACTORS, SUBSTRATE SPECIFICITY, ENZYME REGULATION, BIOPHYSICOCHEMICAL PROPERTIES.
Medline ID
PubMed ID 17676871
Journal Biochemistry
Year 2007
Volume 46
Pages 9840-52
Authors Yip VL, Thompson J, Withers SG
Title Mechanism of GlvA from Bacillus subtilis: a detailed kinetic analysis of a 6-phospho-alpha-glucosidase from glycoside hydrolase family 4.
Related PDB
Related UniProtKB
[10]
Resource
Comments
Medline ID
PubMed ID 18625333
Journal Curr Opin Chem Biol
Year 2008
Volume 12
Pages 532-8
Authors Tanner ME
Title Transient oxidation as a mechanistic strategy in enzymatic catalysis.
Related PDB
Related UniProtKB

Comments
This enzyme belongs to glycosidase family-4, with a retaining mechanism.
This glycosidase family-4 is distinct from other glycosidase families in that it requires NAD cofactor and a divalent metal ion for the reaction, and includes both alpha-glycosidase and beta-glycosidase (see [3], [8], [9] and [10]).
According to the literature [3], [9] and [10], this enzyme catalyzes the following reactions:
(A) Hydride transfer from C3 of substrate to nicotinamide of NAD to form 3-keto intermediate (I00113):
(B) Isomerization from 3-keto intermediate to enediolate intermediate (I00114):
(C) Elimination of D-glucose from C1 atom of the enediolate intermediate, forming enone intermediate (I00115):
(D) Addition of water to C1 atom of the enone intermediate, forming enediolate intermediate (I00116):
(E) Isomerization from enediolate intermediate to 3-keto intermediate (I00117):
(F) Hydride transfer from nicotinamide of NADH to C3 atom of the 3-keto intermediate:
The detailed mechanism seems to be as follows:
(A) Hydride transfer from C3 of substrate to nicotinamide of NAD to form 3-keto intermediate (I00113):
(A0) Manganese ion, which is bound to Cys171, His202 and O7 atom of nicotinamide of NAD, binds O3 and O2 hydroxyl groups, lowering the pKa of O3 hydroxyl group. This facilitates the deprotonation of O3 hydroxyl group.
(A1) Hydride transfer from C3 atom of substrate to nicotinamide of NAD occurs, at the same time as the deprotonation of O3 hydroxyl group, forming 3-keto intermediate (I00113).
(B) Isomerization from 3-keto intermediate to enediolate intermediate (I00114):
(B0) Oxidation of C3 atom acidifies C2 atom, which facilitates the deprotonation of C2 atom. Meanwhile, the manganese ion also facilitate the deprotonation of C2 atom by polarizing carbonyl group at C3. On the other hand, Glu111 modulates the activity of Tyr265 through a water molecule.
(B1) Tyr265 acts as a general base to deprotonate C2 atom, forming an edediolate intermediate (I00114).
(C) Elimination of D-glucose from C1 atom of the enediolate intermediate, forming enone intermediate (I00115):
(C0) The enediolate intermediate, bound to the manganese ion, is reactive.
(C1) Asp172 acts as a general acid to protonate the oxygen atom of the eliminated D-glucose group. This elimination forms an enone intermediate (I00115).
(D) Addition of water to C1 atom of the enone intermediate, forming enediolate intermediate (I00116):
(D0) The enone intermediate (or alpha,beta-unsaturated intermediate), bound to the manganese ion, is reactive for addition at C1.
(D1) Asp172 acts as a general base to deprotonate a water molecule.
(D2) The activated water attacks on the C1 atom of the enone intermediate, forming an enediolate intermediate (I00116).
(E) Isomerization from enediolate intermediate to 3-keto intermediate (I00117):
(E0) O2 and O3 atoms of the enediolate are bound to the manganese ion. On the other hand, Glu111 modulates the activity of Tyr265 through a water molecule.
(E1) Tyr265 acts as a general acid to protonate C2 atom, forming 3-keto intermediate (I00117).
(F) Hydride transfer from nicotinamide of NADH to C3 atom of the 3-keto intermediate:
(F0) Manganese ion, which is bound to Cys171, His202 and O7 atom of nicotinamide of NAD, binds O3 and O2 atoms of the enediolate intermediate.
(F1) Hydride transfer from nicotinamide of NADH to C3 atom of the intermediate occurs, at the same time as the protonation to O3 atom, forming final product.

Created Updated
2009-12-08 2018-04-23