Mandelamide Hydrolase from Pseudomonas putida: Characterization of a New Member of the Amidase Signature Family ² Kota N. Gopalakrishna, ‡ Betty H. Stewart, § Malea M. Kneen, Adriano D. Andricopulo, | George L. Kenyon, and Michael J. McLeish* College of Pharmacy, UniVersity of Michigan, Ann Arbor, Michigan 48109-1065 ReceiVed January 12, 2004; ReVised Manuscript ReceiVed April 14, 2004 ABSTRACT: A recently discovered enzyme in the mandelate pathway of Pseudomonas putida, mandelamide hydrolase (MAH), catalyzes the hydrolysis of mandelamide to mandelic acid and ammonia. Sequence analysis suggests that MAH is a member of the amidase signature family, which is widespread in nature and contains a novel Ser-cis-Ser-Lys catalytic triad. Here we report the expression in Escherichia coli, purification, and characterization of both wild-type and His 6 -tagged MAH. The recombinant enzyme was stable, exhibited a pH optimum of 7.8, and was able to hydrolyze both enantiomers of mandelamide with little enantiospecificity. The His-tagged variant showed no significant change in kinetic constants. Phenylacetamide was found to be the best substrate, with changes in chain length or replacement of the phenyl group producing greatly decreased values of k cat /K m . As with another member of this family, fatty acid amide hydrolase, MAH has the uncommon ability to hydrolyze esters and amides at similar rates. MAH is even more unusual in that it will only hydrolyze esters and amides with little steric bulk. Ethyl and larger esters and N-ethyl and larger amides are not substrates, suggesting that the MAH active site is very sterically hindered. Mutation of each residue in the putative catalytic triad to alanine resulted in total loss of activity for S204A and K100A, while S180A exhibited a 1500-fold decrease in k cat and significant increases in K m values. Overall, the MAH data are similar to those of fatty acid amide hydrolase and support the suggestion that there are two distinct subgroups within the amidase signature family. A number of pseudomonads are able to use one or both of the enantiomers of mandelic acid as their sole carbon source (1). This ability is provided by the enzymes in the mandelate metabolic pathway which facilitate the conversion of both (R)- and (S)-mandelate to benzoate, which is sub- sequently converted to acetyl-CoA and succinyl-CoA by the enzymes of the -ketoadipate pathway (Figure 1). Initial studies indicated that the genes for the mandelate pathway of Pseudomonas putida (ATCC 12633) were all closely clustered on the chromosome (2, 3). Subsequently, the genes were found to reside on a single 10.5 kb restriction fragment (4), and three of the genes, those for mandelate racemase (mdlA), (S)-mandelate dehydrogenase (mdlB), and benzoyl- formate decarboxylase (mdlC), were found to be arranged in an operon (4). Further sequencing of the restriction fragment encoding the enzymes of the mandelate pathway has revealed three more open reading frames which have been denoted mdlD, mdlX, and mdlY(5). The mdlD gene product was shown to be a NAD(P) + -dependent benzaldehyde dehydrogenase, while the deduced amino acid sequence of the mdlX gene product suggested that it encodes a transcriptional regulatory protein (5). The deduced amino acid sequence of the mdlY gene product was considerably homologous with the se- quences of a number of bacterial amide hydrolases (amidases, EC 3.5.1.4). This homology, together with the location of the mdlY gene within the mandelate pathway gene cluster, suggested that mdlY encodes a mandelamide hydrolase. Subsequently, it was demonstrated that the mdlY gene product was indeed able to convert both (R)- and (S)- mandelamide to mandelic acid (5). Mandelamide hydrolase appears to be a member of a large group of enzymes which have been termed the amidase signature (AS) 1 family (6, 7). The family is characterized by the presence of a highly conserved serine- and glycine- rich stretch of approximately 50 amino acids (Figure 2). Members of this family have been identified in more than 90 different organisms (8), including archaea, eubacteria, fungi, nematodes, plants, insects, birds, and mammals (9). Although the biochemical function of the amidases is simply the hydrolysis of the amide bond (RCO-NH 2 ), the biological consequences of the reaction are diverse. These include carbon-nitrogen metabolism in prokaryotes and eukaryotes (10, 11), formation of indole-3-acetic acid in pathogenic plant bacteria (12), degradation of neuromodulatory fatty acid amides in mammals (13, 14), and formation of Gln-tRNAGln ² This research was supported, in part, by U.S. Public Health Service Grant GM-40570 (to G.L.K.). * To whom correspondence should be addressed. Telephone: (734) 615-1787. Fax: (734) 615-3079. E-mail: mcleish@umich.edu. ‡ Current address: Medical College of Georgia, Augusta, GA 30912. § Current address: Austin College, Sherman, TX 75090. | Current address: Center for Structural Molecular Biotechnology, Universidade de Sa ˜o Paulo, Sa ˜o Paulo, Brazil. 1 Abbreviations: AS, amidase signature; MAH, mandelamide hy- drolase; FAAH, fatty acid amide hydrolase; MAE2, malonamidase E2; PAM, peptide amidase; PMSF, phenylmethanesulfonyl fluoride; DFP, diisopropyl fluorophosphate; IPTG, isopropyl -D-thiogalactopyrano- side; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis; WT, wild-type; CD, circular dichroism. 7725 Biochemistry 2004, 43, 7725-7735 10.1021/bi049907q CCC: $27.50 © 2004 American Chemical Society Published on Web 05/25/2004