Mandelamide Hydrolase from Pseudomonas putida:  Characterization of a New Member of the Amidase Signature Family

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.

Specificity and mechanism of mandelamide hydrolase catalysis

“…Protein expression and purification Wild-type and variant MAH were expressed with a Cterminal 6x his-tag using the previously described methods [1,9]. Following purification the enzyme was kept at -20°C in a storage buffer containing 100 mM potassium phosphate buffer, pH 7.8, containing 1 mM EDTA.…”

“…R-Mandelamide was prepared as previously described [1]. Methyl 4-nitrophenylcarbonate (11) was prepared from reaction of 4-nitrophenyl chloroformate with methanol in the presence of triethylamine and recrystallized from an ethyl acetate/hexane mixture (7:3).…”

“…The temperature effect on the hydrolysis of R-mandelamide was measured using the coupled enzyme assay for ammonia [1,9] in 100 mM phosphate buffer at pH 7.8 and containing 1 mM EDTA. The assay mixture comprised the substrate, mandelamide, (10 -200 µM), 250 µM 7 NADH, 800 µM α-ketoglutarate (Sigma), 15 U/mL glutamate dehydrogenase (Sigma), and MAH (0.14 -0.91 µM).…”

“…MAH appears to be associated with the mandelate metabolic pathway of Pseudomonas putida, a pathway that allows this organism to utilize either of the enantiomers of mandelate as the sole carbon source. The gene encoding MAH, located within the gene cluster of the mandelate pathway enzymes of Pseudomonas putida ATCC 12633, has been identified and cloned [1]. MAH belongs to a large group of enzymes termed the amidase signature (AS) family.…”

“…In previous studies, MAH from Pseudomonas putida has been cloned, expressed in E. coli and characterized [1]. The catalytic triad of MAH is composed of Ser204, Ser180, and Lys100.…”