Purification, cloning, and primary structure of an enantiomer-selective amidase from Brevibacterium sp. strain R312: structural evidence for genetic coupling with nitrile hydratase

Abstract

An enantiomer-selective amidase active on several 2-aryl and 2-aryloxy propionamides was identified and purified from Brevibacterium sp. strain R312. Oligonucleotide probes were designed from limited peptide sequence information and were used to clone the corresponding gene, named amdA. Highly significant homologies were found at the amino acid level between the deduced sequence of the enantiomer-selective amidase and the sequences of known amidases such as indoleacetamide hydrolases from Pseudomonas syringae and Agrobacterium tumefaciens and acetamidase from Aspergillus nidulans. Moreover, amdA is found in the same orientation and only 73 bp upstream from the gene coding for nitrile hydratase, strongly suggesting that both genes are part of the same operon. Our results also showed that Rhodococcus sp. strain N-774 and Brevibacterium sp. strain R312 are probably identical, or at least very similar, microorganisms. The characterized amidase is an apparent homodimer of Mr 2 x 54,671 which exhibited under our conditions a specific activity of about 13 to 17 imol of 2-(4-hydroxyphenoxy)propionic R acid formed per min per mg of enzyme from the racemic amide. Large amounts of an active recombinant enzyme could be produced in Escherichia coli at 30TC under the control of an E. coli promoter and ribosome-binding site.

Structure-Based Design of a FAAH Variant That Discriminates between the N-Acyl Ethanolamine and Taurine Families of Signaling Lipids

“…The AS family of hydrolases is found in a wide range of organisms, including archaea (3), eubacteria (1,(4)(5)(6), fungi (7), nematodes, plants, insects, birds (8), and mammals (9,10). Unlike most AS enzymes, FAAH is an integral membrane protein (9,10), which presumably facilitates its role in degrading a large number of amidated lipids in vivo.…”

Comparative Characterization of a Wild Type and Transmembrane Domain-Deleted Fatty Acid Amide Hydrolase: Identification of the Transmembrane Domain as a Site for Oligomerization

“…Fatty acid amide hydrolase (FAAH) 1 (1)(2)(3)(4)(5) is the only identified mammalian member of a family of amidase enzymes known as the "amidase signature" family (6). Despite the presence of these enzymes in prokaryotic (6)(7)(8)(9)(10) and eukaryotic (1,(11)(12)(13) organisms, little is yet known about their catalytic mechanism and structural features.…”

“…Despite the presence of these enzymes in prokaryotic (6)(7)(8)(9)(10) and eukaryotic (1,(11)(12)(13) organisms, little is yet known about their catalytic mechanism and structural features.…”

Enantioselective hydrolysis of racemic naproxen nitrile and naproxen amide to S-naproxen by new bacterial isolates

“…Highly enantioselective amidases have also been observed by other investigators with various a-substituted amides (Kieny L'Homme et al, 1981;Asano et al, 1989;Mayaux et al, 1990Mayaux et al, , 1991Kakeya et al, 1991;Cohen et al, 1992). In general, these activities were found after screening of strains from culture collections or in bacteria which were initially enriched with different often aliphatic nitriles or amides.…”

“…The enzymatic hydrolysis of nitriles represents a very convenient synthetic method for amides and/or carboxylic acids due to the mild reaction conditions. Furthermore, these enzymatic reactions also allow the enantioselective synthesis of optical active amides and carboxylic acids from racemic precursors (Mayaux et al, 1990(Mayaux et al, , 1991Yamamoto et al, 1990;Kakeya et al, 1991;Bianchi et al, 1991;Bhalla et al, 1992;Cohen et al, 1992;Layh et al, 1992).…”