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A novel enzymatic production system of optically pure β-hydroxy α-amino acids was developed. Two enzymes were used for the system: an N-succinyl L-amino acid β-hydroxylase (SadA) belonging to the iron(II)/α-ketoglutarate-dependent dioxygenase superfamily and an N-succinyl L-amino acid desuccinylase (LasA). The genes encoding the two enzymes are part of a gene set responsible for the biosynthesis of peptidyl compounds found in the Burkholderia ambifaria AMMD genome. SadA stereoselectively hydroxylated several N-succinyl aliphatic L-amino acids and produced N-succinyl β-hydroxy L-amino acids, such as N-succinyl- L-β-hydroxyvaline, N-succinyl- L-threonine, (2 S,3 R)- N-succinyl- L-β-hydroxyisoleucine, and N-succinyl- L- threo-β-hydroxyleucine. LasA catalyzed the desuccinylation of various N-succinyl- L-amino acids. Surprisingly, LasA is the first amide bond-forming enzyme belonging to the amidohydrolase superfamily, and has succinylation activity towards the amino group of L-leucine. By combining SadA and LasA in a preparative scale production using N-succinyl- L-leucine as substrate, 2.3 mmol of L- threo-β-hydroxyleucine were successfully produced with 93% conversion and over 99% of diastereomeric excess. Consequently, the new production system described in this study has advantages in optical purity and reaction efficiency for application in the mass production of several β-hydroxy α-amino acids. [ABSTRACT FROM AUTHOR]

N-Carbamoyl-D-amino acid amidohydrolase was purified 119-fold, with 36% overall recovery from a cell-free extract of Comamonas sp. E222c. The purified enzyme was homogeneous as judged by SDS/PAGE. The relative molecular mass of the native enzyme was 120000 and that of the subunit was 40000. The purified enzyme hydrolyzed various N-carbamoyl-D-amino acids to Damino acids, ammonia and carbon dioxide. N-Carbamoyl-D-amino acids having hydrophobic groups served as good substrates for the enzyme. The Km and Vmax values for N-carbamoyl-D-phenylalanine were 19.7 mM and 13.1 units/mg, respectively, and those for N-carbamoyl-D-p-hydroxyphenylglycine were 13.1 mM and 0.56 units/mg, respectively. The enzyme strictly recognized the configuration of the substrate and only the D-enantiomer of the N-carbamoyl amino acid was hydrolyzed. The enzyme activity was not significantly affected by N-carbamoyl-L-amino acids and ammonia. The enzyme was sensitive to thiol reagents and did not require metal ions for its activity. The enzyme did not hydrolyze N-carbamoyl-β-alanine or N-carbamoyl-DL-aspartate suggesting that the enzyme is different from the N-carbamoylamide-hydrolyzing enzymes involved in the pyrimidine degradation pathway. The enzyme did not hydrolyze allantoin and allantoic acid, which are intermediates in purine degradation, N-carbamoylsarcosine and citrulline, suggesting that it is a novel N-carbamoylamide amidohydrolase. [ABSTRACT FROM AUTHOR]

A novel enzyme, arylalkyl acylamidase, which shows a strict specificity for N-acetyl arylalkylamines, but not acetanilide derivatives, was purified from the culture broth of Pseudomonas putida Sc2. The purified enzyme appeared to be homogeneous, as judged by native and SDS/PAGE. The enzyme has a molecular mass of approximately 150 kDa-and consists of four identical subunits. The purified enzyme catalyzed the hydrolysis of N-acetyl-2-phenylethylamine to 2-phenylethylamine and acetic acid at the rate of 6.25 μmol · min-1 · mg-1 at 30°C. It also catalyzed the hydrolysis of various N-acetyl arylalkylamines containing a benzene or indole ring, and acetic acid arylalkyl esters. The enzyme did not hydrolyze acetanilide, N-acetyl aliphatic amines, N-acetyl amino acids, N-acetyl amino sugars or acylthiochotine. The apparent Km for N-acetylbenzylamine, N-acetyl-2-phenyl-ethylamine and N-acetyl-3-phenylpropylamine are 41 mM, 0.31 mM and 1.6 mM, respectively. The purified enzyme was sensitive to thiol reagents such as Ag2SO4, HgCl2 and p-chloromercuribenzoic acid, and its activity was enhanced by divalent metal ions such as Zn2+, Mg2+ and Mn2+. [ABSTRACT FROM AUTHOR]