Publications

• Production of R-(-)-mandelic acid from mandelonitrile by Alcaligenes faecalis ATCC 8750.

  Yamamoto K., Oishi K., Fujimatsu I., Komatsu K.

  R-(-)-Mandelic acid was produced from racemic mandelonitrile by Alcaligenes faecalis ATCC 8750. Ammonium acetate or L-glutamic acid as the carbon source and n-butyronitrile as the inducer in the culture medium were effective for bacterial growth and the induction of R-(-)-mandelic acid-producing a ... >> More

  R-(-)-Mandelic acid was produced from racemic mandelonitrile by Alcaligenes faecalis ATCC 8750. Ammonium acetate or L-glutamic acid as the carbon source and n-butyronitrile as the inducer in the culture medium were effective for bacterial growth and the induction of R-(-)-mandelic acid-producing activity. The R-(-)-mandelic acid formed from mandelonitrile by resting cells was present in a 100% enantiomeric excess. A. faecalis ATCC 8750 has an R-enantioselective nitrilase for mandelonitrile and an amidase for mandelamide. As R-(-)-mandelic acid was produced from racemic mandelonitrile in a yield of 91%, whereas no S-mandelonitrile was left, the S-mandelonitrile remaining in the reaction is spontaneously racemized because of the chemical equilibrium and is used as the substrate. Consequently, almost all the mandelonitrile is consumed and converted to R-(-)-mandelic acid. R-(-)-Mandelic acid was also produced when benzaldehyde plus HCN was used as the substrate. << Less

  Appl Environ Microbiol 57:3028-3032(1991) [PubMed] [EuropePMC]

• Mandelamide hydrolase from Pseudomonas putida: characterization of a new member of the amidase signature family.

  Gopalakrishna K.N., Stewart B.H., Kneen M.M., Andricopulo A.D., Kenyon G.L., McLeish M.J.

  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 contain ... >> More

  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. << Less

  Biochemistry 43:7725-7735(2004) [PubMed] [EuropePMC]

• Identification and characterization of a mandelamide hydrolase and an NAD(P)+-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633.

  McLeish M.J., Kneen M.M., Gopalakrishna K.N., Koo C.W., Babbitt P.C., Gerlt J.A., Kenyon G.L.

  The enzymes of the mandelate metabolic pathway permit Pseudomonas putida ATCC 12633 to utilize either or both enantiomers of mandelate as the sole carbon source. The genes encoding the mandelate pathway were found to lie on a single 10.5-kb restriction fragment. Part of that fragment was shown to ... >> More

  The enzymes of the mandelate metabolic pathway permit Pseudomonas putida ATCC 12633 to utilize either or both enantiomers of mandelate as the sole carbon source. The genes encoding the mandelate pathway were found to lie on a single 10.5-kb restriction fragment. Part of that fragment was shown to contain the genes coding for mandelate racemase, mandelate dehydrogenase, and benzoylformate decarboxylase arranged in an operon. Here we report the sequencing of the remainder of the restriction fragment, which revealed three further open reading frames, denoted mdlX, mdlY, and mdlD. All were transcribed in the opposite direction from the genes of the mdlABC operon. Sequence alignments suggested that the open reading frames encoded a regulatory protein (mdlX), a member of the amidase signature family (mdlY), and an NAD(P)(+)-dependent dehydrogenase (mdlD). The mdlY and mdlD genes were isolated and expressed in Escherichia coli, and the purified gene products were characterized as a mandelamide hydrolase and an NAD(P)(+)-dependent benzaldehyde dehydrogenase, respectively. << Less

  J. Bacteriol. 185:2451-2456(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Using directed evolution to probe the substrate specificity of mandelamide hydrolase.

  Wang P.F., Yep A., Kenyon G.L., McLeish M.J.

  Mandelamide hydrolase (MAH), a member of the amidase signature family, catalyzes the hydrolysis of mandelamide to mandelate and ammonia. X-ray structures of several members of this family, but not that of MAH, have been reported. These reveal nearly superimposable conformations of the unusual Ser- ... >> More

  Mandelamide hydrolase (MAH), a member of the amidase signature family, catalyzes the hydrolysis of mandelamide to mandelate and ammonia. X-ray structures of several members of this family, but not that of MAH, have been reported. These reveal nearly superimposable conformations of the unusual Ser-cisSer-Lys catalytic triad. Conversely, the residues involved in substrate recognition are not conserved, implying that the binding pocket could be modified to change the substrate specificity, perhaps by directed evolution. Here we show that MAH is able to hydrolyze small aliphatic substrates such as lactamide, albeit with low efficiency. A selection method to monitor changes in mandelamide/lactamide preference was developed and used to identify several mutations affecting substrate binding. A homology model places some of these mutations close to the catalytic triad, presumably in the MAH active site. In particular, Gly202 appears to control the preference for aromatic substrates as the G202A variant showed three orders of magnitude decrease in k(cat)/K(m) for (R)- and (S)-mandelamide. This reduction in activity increased to six orders of magnitude for the G202V variant. << Less

  Protein Eng. Des. Sel. 22:103-110(2009) [PubMed] [EuropePMC]