Isolation and molecular characterization of a novel d‐hydantoinase from Jannaschia sp. CCS1

Abstract

Hydantoinases (HYDs) are important enzymes for industrial production of optically pure amino acids, which are widely used as precursors for various semi‐synthetic antibiotics. By a process coupling genomic data mining with activity screening, a new hydantoinase, tentatively designated HYDJs, was identified from Jannaschia sp. CCS1 and overexpressed in Escherichia coli. The specific activity of HYDJs on d,l‐p‐hydroxyphenylhydantoin as the substrate was three times higher than that of the hydantoinase originating from Burkholderia pickettii (HYDBp) that is currently used in industry. The enzyme obtained was a homotetramer with a molecular mass of 253 kDa. The pH and temperature optima for HYDJs were 7.6 and 50 °C respectively, similar to those of HYDBp. Kinetic analysis showed that HYDJs has a higher kcat value on d,l‐p‐hydroxyphenylhydantoin than HYDBp does. Homology modeling and substrate docking analyses of HYDJs and HYDBp were performed, and the results revealed an enlarged substrate binding pocket in HYDJs, which may allow better access of substrates to the catalytic centre and could account for the increased specific activity of HYDJs. Three amino acid residues critical for HYDJs activity, Phe63, Leu92 and Phe150 were also identified by substrate docking and site‐directed mutagenesis. Application of this high‐specific activity HYDJs could improve the industrial production of optically pure amino acids, such as d‐p‐hydroxyphenylglycine. Moreover, the structural analysis also provides new insights on enzyme–substrate interaction, which shed light on engineering of hydantoinases for high catalytic activity.

Stereoselective hydrolysis of aryl-substituted dihydropyrimidines by hydantoinases

“…The hydantoinase process has a theoretical yield of 100% and is industrially applied for the biocatalytic synthesis of enantiopure α-amino acids, e.g., α-D-phenylglycine and α-D-p-hydroxyphenylglycine (Martínez-Gómez et al 2007;Cai et al 2009). For this purpose, a hydantoin racemase, an enantioselective hydantoinase, and an enantioselective N-carbamoyl-α-amino acid hydrolase are employed to produce optically pure α-amino acids starting from racemic hydantoins (Fig.…”

Homologous Recombination in Core Genomes Facilitates Marine Bacterial Adaptation

“…One example is aminoglycoside phosphotransferase (PGA1_c00530), which catalyzes the phosphorylation of the aminoglycoside and macrolide antibiotics, leading to their inactivation and to bacterial antibiotic resistance (48). A second example is D-hydantoinase (PGA1_c12850), which catalyzes the hydrolysis of 5-substituted hydantoins in microorganisms, leading to enantiomerically pure N-carbamyl amino acids, and is widely used for the production of antibiotics, peptide hormones, pyrethroids, and pesticides (49). A third example is the deacylase PagL (PGA1_c04940), which is important for virulence and resistance to cationic antimicrobial peptides via regulating lipid A modification (50).…”

Toward a cell-free hydantoinase process: screening for expression optimization and one-step purification as well as immobilization of hydantoinase and carbamoylase

“…Though, even the chaperone set C2 with the best results concerning specific activities is only half as active as the d -Hyd(co) without coexpression of chaperones with same cultivation conditions. Despite the fact that the coexpression of chaperones had been applied successfully in many cases and even for hydantoinases (Cai et al 2009), it has not been effective in every case. Many possible reasons have been discussed, like enhanced proteolytic activities upon overexpression of chaperones (Straus et al 1990; Kandror et al 1994, 1999; Kondo and Nishihara 2000; Nishihara et al 2000; García-Fruitós et al 2007; Zahrl et al 2007).…”