Gene Identification and Characterization of the Pyridoxine Degradative Enzyme .ALPHA.-(N-Acetylaminomethylene)succinic Acid Amidohydrolase from Mesorhizobium loti MAFF303099

Abstract

SummaryWe have found for the first time that a chromosomal gene, mlr6787, in Mesorhizobium loti encodes the pyridoxine degradative enzyme ␣ -( N -acetylaminomethylene)succinic acid (AAMS) amidohydrolase. The recombinant enzyme expressed in Escherichia coli cells was homogeneously purified and characterized. The enzyme consisted of two subunits each with a molecular mass of 34,000 Ϯ 1,000 Da, and exhibited Km and kcat values of 53.7 Ϯ 6 M and 307.3 Ϯ 12 min Ϫ 1 , respectively. The enzyme required no cofactor or metal ion. The primary structure of AAMS amidohydrolase was elucidated for the first time here. The primary structure of the enzyme protein showed no significant identity to those of known hydrolase proteins and low homology to those of fluoroacetate dehalogenase (PDB code, 1Y37), haloalkane dehalogenase (1K5P), and aryl esterase (1VA4).

Structure Determination and Characterization of the Vitamin B6 Degradative Enzyme (E)-2-(Acetamidomethylene)succinate Hydrolase,

“…The activity of the active site mutants S106A, D130N, and S230C were assayed by measuring the decrease in the absorbance at 261 nm, the λ max for E -2AMS as previously described (7, 10). Each of these mutants was found to be completely inactive relative to the native enzyme, which retained an activity comparable to published results.…”

“…This family has been structurally well characterized and includes esterases, lipases, epoxidases, alkane dehalogenases, carbon-carbon bond hydrolases, amidohydrolases, and thioesterases (33, 34). Previous analysis of the primary sequence for E -2AMS hydrolase using BLAST suggested that this enzyme was a member of the α/β hydrolase superfamily, despite a low sequence identity to this family of enzymes (10). A DALI search was performed using the structure of E -2AMS hydrolase and these results confirmed that E -2AMS hydrolase is a member of the α/β hydrolase family.…”

“…This enzyme catalyzes the hydrolysis of E -2AMS to succinic semialdehyde, ammonia, acetate, and carbon dioxide. The products of this reaction have been conclusively identified and the steady state kinetics of the enzyme have been characterized (7, 10). A similar reaction in nicotinate catabolism, catalyzing the amide hydrolysis of 2-(enamine)-glutarate, is performed by an enamidase, a member of the amidohydrolase superfamily (15).…”

“…This superfamily often utilizes a catalytic triad consisting of an active site nucleophile, an absolutely conserved histidine residue, and an acidic residue. Sequence alignments failed to identify this acidic residue and it was unclear how this enzyme catalyzed its metal-independent hydrolysis reaction (10). The structure reported here identifies this acidic residue and also suggests several other residues within the active site that could be involved in substrate binding and catalysis.…”

How Does (E)-2-(Acetamidomethylene)succinate Bind to Its Hydrolase? From the Binding Process to the Final Result

“…The hydrolysis reaction has been well-characterized and the corresponding steady-state kinetic parameters have also been determined [13], [14]. Recently McCulloch and co-workers have proved that the hydrolase only utilizes E -2AMS rather than Z -2AMS as its substrate [13].…”