Biomedical subjects

Sachio Herai

Publications and source records attributed to Sachio Herai.

4 recordsLinked to original sources

PubMed2005

High-level expression of a novel amine-synthesizing enzyme, N-substituted formamide deformylase, in Streptomyces with a strong protein expression system.

N-substituted formamide deformylase (NfdA) from Arthrobacter pascens F164 is a novel deformylase involved in the metabolism of isonitriles. The enzyme catalyzes the deformylation of an N-substituted formamide, which is produced from the corresponding isonitrile, to yield the corresponding amine and formate. The nfdA gene from A. pascens F164 was cloned into different types of expression vectors for Escherichia coli and Streptomyces strains. Expression in E. coli resulted in the accumulation of an insoluble protein. However, Streptomyces strains transformed with a P(nitA)-NitR system, which we very recently developed as a regulatory gene expression system for streptomycetes, allowed the heterologous overproduction of NfdA in an active form. When Streptomyces lividans TK24 transformed with pSH19-nfdA was cultured under the optimum conditions, the NfdA activity of the cell-free extract amounted to 8.5 U/mg, which was 29-fold higher than that of A. pascens F164. The enzyme also comprised approximately 20% of the total extractable cellular protein. The recombinant enzyme was purified to homogeneity and characterized. The expression system established here will allow structural analysis and mutagenesis studies of NfdA.

Amidohydrolases↗

PubMed2004

Hyper-inducible expression system for streptomycetes.

Streptomycetes produce useful enzymes and a wide variety of secondary metabolites with potent biological activities (e.g., antibiotics, immunosuppressors, pesticides, etc.). Despite their importance in the pharmaceutical and agrochemical fields, there have been no reports for practical expression systems in streptomycetes. Here, we developed a "P(nitA)-NitR" system for regulatory gene expression in streptomycetes based on the expression mechanism of Rhodococcus rhodochrous J1 nitrilase, which is highly induced by an inexpensive and safe inducer, epsilon-caprolactam. Heterologous protein expression experiments demonstrated that the system allowed suppressed basal expression and hyper-inducible expression, yielding target protein levels of as high as approximately 40% of all soluble protein. Furthermore, the system functioned in important streptomycete strains. Thus, the P(nitA)-NitR system should be a powerful tool for improving the productivity of various useful products in streptomycetes.

Aminohydrolases↗

PubMed2002

Isonitrile hydratase from Pseudomonas putida N19-2. Cloning, sequencing, gene expression, and identification of its active acid residue.

Isonitrile hydratase is a novel enzyme in Pseudomonas putida N19-2 that catalyzes the conversion of isonitriles to N-substituted formamides. Based on N-terminal and internal amino acid sequences, a 535-bp DNA fragment corresponding to a portion of the isonitrile hydratase gene was amplified, which was used as a probe to clone a 6.4-kb DNA fragment containing the whole gene. Sequence analysis of the 6.4-kb fragment revealed that the isonitrile hydratase gene (inhA) was 684 nucleotides long and encoded a protein with a molecular mass of 24,211 Da. Overexpression of inhA in Escherichia coli gave a large amount of soluble isonitrile hydratase exhibiting the same molecular and catalytic properties as the native enzyme from the Pseudomonas strain. The predicted amino acid sequence of inhA showed low similarity to that of an intracellular protease in Pyrococcus horikoshii (PH1704), and an active cysteine residue in the protease was conserved in the isonitrile hydratase at the corresponding position (Cys-101). A mutant enzyme containing Ala instead of Cys-101 did not exhibit isonitrile hydratase activity at all, demonstrating the essential role of this residue in the catalytic function.

Amino Acid Sequence↗

PubMed2002

Site-directed mutagenesis for cysteine residues of cobalt-containing nitrile hydratase.

Three cysteine residues, which are completely conserved among alpha-subunits in all nitrile hydratases, are thought to be the ligands of a metal ion in the catalytic center of this enzyme. These cysteine residues (i.e. alpha C102, alpha C105 and alpha C107) in the high-molecular-mass nitrile hydratase (H-NHase) of Rhodococcus rhodochrous J1 were replaced with alanine by site-directed mutagenesis using the R. rhodochrous ATCC12674 host-vector system, and the resultant transformants were investigated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the cell-free extracts of each mutant transformant revealed that four mutant transformants (i.e. alpha C105A, alpha C107A, alpha C102A/C105A and alpha C105A/C107A) showed predominant alpha- and beta-subunit protein bands with a mobility identical to those of the native H-NHase, while three mutant transformants (i.e. alpha C102A, alpha C102A/C107A and alpha C102A/C105A/C107A) did not produce the corresponding proteins. The purified former four mutant enzymes showed neither enzymatic activity nor the maximum absorption at 410 nm which was detected in the wild type H-NHase. They also did not contain cobalt ions. Based upon these findings, these three cysteine residues were found to be essential for the active expression of H-NHase.

Amino Acid Sequence↗