Insights into the mechanism of catalysis by the P-C bond-cleaving enzyme phosphonoacetaldehyde hydrolase derived from gene sequence analysis and mutagenesis.
Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and inorganic phosphate. In this study, the genes encoding phosphonatase in Bacillus cereus and in Salmonella typhimurium were cloned for high-level expression in Escherichia coli. The kinetic properties of the purified, recombinant phosphonatases were determined. The Schiff base mechanism known to operate in the B. cereus enzyme was verified for the S. typhimurium enzyme by phosphonoacetaldehyde-sodium borohydride-induced inactivation and by site-directed mutagenesis of the catalytic lysine 53. The protein sequence inferred from the B. cereus phosphonatase gene was determined, and this sequence was used along with that from the S. typhimurium phosphonatase gene sequence to search the primary sequence databases for possible structural homologues. We found that phosphonatase belongs to a novel family of hydrolases which appear to use a highly conserved active site aspartate residue in covalent catalysis. On the basis of this finding and the known stereochemical course of phosphonatase-catalyzed hydrolysis at phosphorus (retention), we propose a mechanism which involves Schiff base formation with lysine 53 followed by phosphoryl transfer to aspartate (at position 11 in the S. typhimurium enzyme and position 12 in the B. cereusphosphonatase) and last hydrolysis at the imine C(1) and acyl phosphate phosphorus.
Baker
A S
AS
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742, USA.
Ciocci
M J
MJ
Metcalf
W W
WW
Kim
J
J
Babbitt
P C
PC
Wanner
B L
BL
Martin
B M
BM
Dunaway-Mariano
D
D
D000595:Amino Acid Sequence; D001409:Bacillus cereus / Q000201:enzymology / Q000235:genetics; D001665:Binding Sites; D002244:Carbon / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D019143:Evolution, Molecular; D015964:Gene Expression Regulation, Bacterial; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D010758:Phosphorus / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics; D012545:Schiff Bases / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D012545:Schiff Bases; D010758:Phosphorus; D002244:Carbon; D006867:Hydrolases; C023920:phosphonoacetaldehyde hydrolase; D008239:Lysine