Cloning and sequencing of the genes involved in the conversion of 5-substituted hydantoins to the corresponding L-amino acids from the native plasmid of Pseudomonas sp. strain NS671

Abstract

Pseudomonas sp. strain NS671, which produces L-amino acids asymmetrically from the corresponding racemic 5-substituted hydantoins, harbored a plasmid of 172 kb. Curing experiments suggest that this plasmid, designated pHN671, is responsible for the conversion of 5-substituted hydantoins to their corresponding L-amino acids by strain NS671. DNA fragments containing the genes involved in this conversion were cloned from pHN671 in Escherichia coli by using pUC18 as a cloning vector. The smaDlest recombinant plasmid, designated pHPB12, contained a 7.5-kb insert DNA. The nucleotide sequence of the insert DNA was determined, and three closely spaced open reading frames predicted to encode peptides with molecular masses of 75.6, 64.9, and 45.7 kDa were found. These open reading frames were designated hyuA, hyuB, and hyuC, respectively. Cell extracts from E. coli carrying deletion derivatives of pHPB12 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the gene products of hyuA, hyuB, and hyuC were identified. The functions of these gene products were also examined with the deletion derivatives. The results indicate that both hyuA and hyuB are involved in the conversions of D-and L-5-substituted hydantoins to corresponding N-carbamyl-D-and N-carbamyl-L-amino acids, respectively, and that hyuC is involved in the conversion of N-carbamyl-L-amino acids to L-amino acids.Racemic 5-substituted hydantoins are intermediates in the conventional chemical synthesis of the corresponding racemic amino acids. It has been reported that some bacteria belonging to the genera Bacillus (33,35,37), Flavobacterium (22,27), and Arthrobacter (30), etc., can convert racemic 5-substituted hydantoins to the corresponding L-amino acids. The bacterial conversion consists of the following three successive steps (22,30,31,35,36,39). The first step is either spontaneous or the enzymatic racemization of 5-substituted hydantoins. The second step is a ring-opening hydrolysis of 5-substituted hydantoins with an enzyme called hydantoinase, and corresponding N-carbamyl-amino acids are produced. This step is thought to be either L-isomer specific or nonspecific but reversible. In the third step, N-carbamyl-L-amino acids are hydrolyzed to L-amino acids with an L-isomer-specific enzyme. As a result, the whole racemic 5-substituted hydantoins are converted to corresponding L-amino acids.Pseudomonas sp. strain NS671, which produces L-amino acids asymmetrically from corresponding racemic 5-substituted hydantoins, is one of the bacteria isolated in our laboratory from soil (14). T o investigate the molecular mechanisms of the reactions catalyzed by strain NS671, we attempted to clone the genes involved in the conversion of 5-substituted hydantoins to corresponding L-amino acids. In this paper, we describe the cloning and sequencing of the genes and examine the functions of these gene products. MATERIALS AND METHODSBacterial strains and plasmids. Bacterial strain NS671 (FERM P-9543, Fermentation Research Institute, Agency of Industria...

Molecular Cloning and Expression of thehyuGenes fromMicrobacterium liquefaciensAJ 3912, Responsible for the Conversion of 5-Substituted Hydantoins to α-Amino Acids, inEscherichia coli

“…Such hyu gene clusters have also been observed in Pseudomonas sp. NS761, 17,18) A. aurescens DSM 3747, 19) and Agrobacterium sp. IP I-671.…”

Purification and Characterization of an ATP‐dependent Amidohydrolase, N‐methylhydantoin Amidohydrolase, from Pseudomonas putida 77

“…However, a DNA fragment containing the gene of the non-stereospecific hydantoin amidohydrolase of Pseudomonus sp. NS671 was cloned and analyzed [40]. The enzyme consists of two different subunits; the large subunit and the small subunit of the enzyme are encoded in different open reading frames, close together, and are translationally coupled.…”

Identification, Cloning, and Sequencing of the Genes Involved in the Conversion ofD,L-2-Amino-Δ2-Thiazoline-4-Carboxylic Acid to …

“…These are the enzymes from A. aurescence DSM3747, 16) B. stearothermophilus NS714A, 17) Pseudomonas sp. NS671, 18) B. subtilis (GenBank accession number Z99120), E. coli (U82664), and H. in‰uenzae (U32740). The enzymes from A. aurescence, B. stearothermophilus and Pseudomonas sp.…”

“…However, ATC hydrolase had no signiˆcant similarity to hydantoinases from Pseudomonas sp. NS671 18) and P. putida, 27) and crude extracts from the transformant expressing ATC hydrolase activity did not hydrolyze hydantoins (data not shown). What kind of substrates except for ATC would be hydrolyzed by ATC hydrolase?…”

“…18,22,23) Several microorganisms producing N-carbamoyl-D-and W or N-carbamoyl-Lamino acid amidohydrolases have been isolated, and some of these enzymes were puriˆed and characterized. 16,19,21,[24][25][26] These results suggest that there are 2 classes of N-carbamoyl amino acid amidohydrolases, N-carbamoyl-D-and N-carbamoyl-L-amino acid amidohydrolases.…”