DB code: D00827

RLCP classification	9.1050.192630.8500 : Hydride transfer
	8.131.704320.8015 : Isomerization
	5.1020.2074300.8301 : Elimination
	4.1010.695600.8301 : Addition
	8.113.904850.8015 : Isomerization
	9.5010.194230.8500 : Hydride transfer
CATH domain	3.40.50.720 : Rossmann fold
CATH domain	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)
3.40.50.720 : 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
3.90.110.10 : L-2-Hydroxyisocaproate Dehydrogenase; Chain A, domain 2	D00005 D00008 M00171

CATH domain

Related DB codes (homologues)

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

3.90.110.10 : L-2-Hydroxyisocaproate Dehydrogenase; Chain A, domain 2

D00005 D00008 M00171

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 18291 35154	15846 15846	15703 15703	15377 15377	4167 4167	17665 17665
PubChem	23930 23930	5893 5893	439874 439874	22247451 962 22247451 962	5793 5793	439284 439284

1nrhX01	Unbound	Bound:NAD	Unbound		Unbound	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound
1u8xX01	Unbound	Bound:NAD	Unbound		Unbound	Unbound	Unbound	Unbound	Unbound	Unbound	Unbound
1nrhX02	Bound:_MN	Unbound	Unbound		Unbound	Analogue:G6P	Unbound	Unbound	Unbound	Unbound	Unbound
1u8xX02	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	GLU 111
1u8xX01	GLU 111
1nrhX02	ASP 172;TYR 265	CYS 171;HIS 202 (Manganese binding)
1u8xX02	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	24i1-2)
Pages	167-176
Authors	Vivian L. Y. Yip; Stephen G. Withers
Title	Family 4 glycoside hydrolases are special: The first À-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
(A) Hydride transfer from C3 of substrate to nicotinamide of NAD to form 3-keto intermediate (I00113): 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: (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.

Comments

(A) Hydride transfer from C3 of substrate to nicotinamide of NAD to form 3-keto intermediate (I00113):
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:
(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	2011-12-27