The Crystal Structure of Bacillus cereus Phosphonoacetaldehyde Hydrolase: Insight into Catalysis of Phosphorus Bond Cleavage and Catalytic Diversification within the HAD Enzyme Superfamily
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- 作者：Marc C. Morais ; Wenhai Zhang ; Angela S. Baker ; Guofeng Zhang ; Debra Dunaway-Mariano ; and Karen N. Allen
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：August 29, 2000
- 年：2000
- 卷：39
- 期：34
- 页码：10385 - 10396
- 全文大小：846K
- 年卷期：v.39,no.34(August 29, 2000)
- ISSN：1520-4995
文摘
Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and phosphate using Mg(II) as cofactor. The reaction proceeds via a novelbicovalent catalytic mechanism in which an active-site nucleophile abstracts the phosphoryl group fromthe Schiff-base intermediate formed from Lys53 and phosphonoacetaldehyde. In this study, the X-raycrystal structure of the Bacillus cereus phosphonatase homodimer complexed with the phosphate (product)analogue tungstate (Ki = 50 
M) and the Mg(II) cofactor was determined to 3.0 Å resolution with anRcryst = 0.248 and Rfree = 0.284. Each monomer is made up of an 
/
core domain consisting of a centrallylocated six-stranded parallel -sheet surrounded by six -helices. Two flexible, solvated linkers connectto a small cap domain (residues 21-99) that consists of an antiparallel, five-helix bundle. The subunit-subunit interface, formed by the symmetrical packing of the two 8 helices from the respective coredomains, is stabilized through the hydrophobic effect derived from the desolvation of paired Met171,Trp164, Tyr162, Tyr167, and Tyr176 side chains. The active site is located at the domain-domain interfaceof each subunit. The Schiff base forming Lys53 is positioned on the cap domain while tungstate andMg(II) are bound to the core domain. Mg(II) ligands include two oxygens of the tungstate ligand, oneoxygen of the carboxylates of Asp12 and Asp186, the backbone carbonyl oxygen of Ala14, and a waterthat forms a hydrogen bond with the carboxylate of Asp190 and Thr187. The guanidinium group ofArg160 binds tungstate and the proposed nucleophile Asp12, which is suitably positioned for in-lineattack at the tungsten atom. The side chains of the core domain residue Tyr128 and the cap domainresidues Cys22 and Lys53 are located nearby. The identity of Asp12 as the active-site nucleophile wasfurther evidenced by the observed removal of catalytic activity resulting from Asp12Ala substitution.The similarity of backbone folds observed in phosphonatase and the 2-haloacid dehalogenase of the HADenzyme superfamily indicated common ancestry. Superposition of the two structures revealed a conservedactive-site scaffold having distinct catalytic stations. Analysis of the usage of polar amino acid residuesat these stations by the dehalogenases, phosphonatases, phosphatases, and phosphomutases of the HADsuperfamily suggests possible ways in which the active site of an ancient enzyme ancestor might havebeen diversified for catalysis of C-X, P-C, and P-O bond cleavage reactions.
M) and the Mg(II) cofactor was determined to 3.0 Å resolution with anRcryst = 0.248 and Rfree = 0.284. Each monomer is made up of an / core domain consisting of a centrallylocated six-stranded parallel -sheet surrounded by six -helices. Two flexible, solvated linkers connectto a small cap domain (residues 21-99) that consists of an antiparallel, five-helix bundle. The subunit-subunit interface, formed by the symmetrical packing of the two 8 helices from the respective coredomains, is stabilized through the hydrophobic effect derived from the desolvation of paired Met171,Trp164, Tyr162, Tyr167, and Tyr176 side chains. The active site is located at the domain-domain interfaceof each subunit. The Schiff base forming Lys53 is positioned on the cap domain while tungstate andMg(II) are bound to the core domain. Mg(II) ligands include two oxygens of the tungstate ligand, oneoxygen of the carboxylates of Asp12 and Asp186, the backbone carbonyl oxygen of Ala14, and a waterthat forms a hydrogen bond with the carboxylate of Asp190 and Thr187. The guanidinium group ofArg160 binds tungstate and the proposed nucleophile Asp12, which is suitably positioned for in-lineattack at the tungsten atom. The side chains of the core domain residue Tyr128 and the cap domainresidues Cys22 and Lys53 are located nearby. The identity of Asp12 as the active-site nucleophile wasfurther evidenced by the observed removal of catalytic activity resulting from Asp12Ala substitution.The similarity of backbone folds observed in phosphonatase and the 2-haloacid dehalogenase of the HADenzyme superfamily indicated common ancestry. Superposition of the two structures revealed a conservedactive-site scaffold having distinct catalytic stations. Analysis of the usage of polar amino acid residuesat these stations by the dehalogenases, phosphonatases, phosphatases, and phosphomutases of the HADsuperfamily suggests possible ways in which the active site of an ancient enzyme ancestor might havebeen diversified for catalysis of C-X, P-C, and P-O bond cleavage reactions.