Cloning and Heterologous Expression of an Enantioselective Amidase from Rhodococcus erythropolis Strain MP50

Abstract

The gene for an enantioselective amidase was cloned from Rhodococcus erythropolis MP50, which utilizes various aromatic nitriles via a nitrile hydratase/amidase system as nitrogen sources. The gene encoded a protein of 525 amino acids which corresponded to a protein with a molecular mass of 55.5 kDa. The deduced complete amino acid sequence showed homology to other enantioselective amidases from different bacterial genera. The nucleotide sequence approximately 2.5 kb upstream and downstream of the amidase gene was determined, but no indications for a structural coupling of the amidase gene with the genes for a nitrile hydratase were found. The amidase gene was carried by an approximately 40-kb circular plasmid in R. erythropolis MP50. The amidase was heterologously expressed in Escherichia coli and shown to hydrolyze 2-phenylpropionamide, ␣-chlorophenylacetamide, and ␣-methoxyphenylacetamide with high enantioselectivity; mandeloamide and 2-methyl-3-phenylpropionamide were also converted, but only with reduced enantioselectivity. The recombinant E. coli strain which synthesized the amidase gene was shown to grow with organic amides as nitrogen sources. A comparison of the amidase activities observed with whole cells or cell extracts of the recombinant E. coli strain suggested that the transport of the amides into the cells becomes the rate-limiting step for amide hydrolysis in recombinant E. coli strains.Acylamide amidohydrolases (amidases) are used in biocatalysis for the chemoselective, regioselective, or enantioselective hydrolysis of various amides (17, 59). The chemo-and regioselectivities of amidases are utilized for the production of antibiotics (penicillin acylase), the hydrolysis of C-terminal amide groups in peptides (peptide amidase), the analysis of glycoproteins [peptide-N 4 -(N-acetyl-␤-D-glucosaminyl)asparagine amidase F], or the transformation of cyclic imides (halfamidase, imidase) (5,25,50,55,57,59). Enantioselective amidases are used for the production of optical active D-or L-␣-amino acids, hydroxycarboxylic acids, or ␣-methylarylacetic and ␣-methoxyarylacetic acids. L-specific aminoamidases have been reported for Pseudomonas putida, Mycobacterium neoaurum, and Stenotrophomonas maltophilia, and a D-specific amino acid amidase has been found in Ochrobactrum anthropi. These enzymes usually also convert certain peptides and are therefore referred to as aminopeptidases (3,21,22,41).An evolutionarily different group of amidases has been found which enantioselectively converts 2-methylphenylacetamide (2-phenylpropionamide) and other ␣-methylarylacetamides. This group of amidases has been found in different rhodococci but also in gram-negative organisms, such as Pseudomonas chlororaphis B23 or Agrobacterium tumefaciens d3 (6,31,44,45,49).One of the best-characterized amidases with the ability to enantioselectively hydrolyze various ␣-methylarylacetamides that has been described is from Rhodococcus erythropolis MP50. This isolate was obtained from an enrichment with naproxen nitrile as sole nitrogen s...

The Novel BacterialN-Demethylase PdmAB Is Responsible for the Initial Step ofN,N-Dimethyl-Substituted Phenylurea Herbicide Degradation

“…This was further supported by the observation that tpnX was sandwiched between two identical inverted repeats of 16 bp (5=-GGCATTGT CAAGTTGC-3=) (Fig. 2C), which were very similar to the inverted repeats described for ISRh1 (60,61). orf1 encodes a protein consisting of 281 amino acid residues, showing moderate sequence identity with 2,3-dihydroxy-2,3-dihydrophenylpropionate dehydrogenase.…”

TraA is required for megaplasmid conjugation in Rhodococcus erythropolis AN12

“…It is well known that double-stranded DNA breaks can trigger homologous recombination at the site of the lesion (Cox, 1999;van den Bosch et al, 2002). This phenomenon was Wrst exploited to achieve genetic transformations of Saccharomyces cerevisiae (OrrWeaver et al, 1981).…”

“…Gene clusters that enable these unique enzymatic pathways often reside on large episomal elements called megaplasmids. Rhodococcus megaplasmids exhibit either circular or linear topology, and range from less than 50 kb to greater than one megabasepairs in length (Priefert et al, 2004;Shimizu et al, 2001;Stecker et al, 2003;Trott et al, 2002). Though megaplasmids are common within this genus of actinomycetes, this type of extrachromosomal replicon is not exclusive to rhodococci.…”

Hydrolysis of Amides

“…Other chiral substrates used in amidase resolutions are 2-(4-chlorophenyl)propionamide (12b) [86], 2-(4-methoxyphenyl)propionamide (12c) [86,96], 2-(4-chlorophenyl)-3-methylbutyramide (16) [76,79,83], 2-phenyl-4-pentenoic acid amide (17c R ¼ allyl) [116], 4-cyano-2-phenylbutyramide (18) namide (19) [25,45,48,49] [(R)-selective hydrolysis; absolute selectivity in line with the (S)-selectivity for 2-arylpropionamides], atrolactic acid amide (20) [93], lysergamide (21) [7,87], 2-methyl-3-phenylpropionamide (22) [32,90], 2-hydroxy-4-phenylbutyramide (23 R ¼ H) and 2-hydroxy-2-methyl-4-phenylbutyramide (24 R ¼ Me)) [93], 2-chloro-phenylacetic acid amide (25) [32,90], mandelic acid amide (26) [90,93], O-methyl-mandelic acid amide (27) [32,90], lactic acid amide (28) [59], 4-chloro-3-hydroxybutyramide (29) [16], carnitine amide (30) [107][108][109], 3-benzoyloxypentanoic acid amide (31) [96], 2-benzyl-2-methylmalondiamide (32) [96], and 2-butyl-2-methylmalondiamide (33) [95]. The hydrolysis of a-and b-amino acid amides, cyclic...…”

“…Various other amidases described in Table 15.1 have been cloned and overexpressed in Escherichia coli or Brevibacterium lactofermentum strains [16,25,65,90,98,106].…”