Development of a novel single-chain l-glutamate oxidase from Streptomyces sp. X-119-6 by inserting flexible linkers

Hiroki Yamaguchi; Kazutoshi Takahashi; Moemi Tatsumi; Uno Tagami; Toshimi Mizukoshi; Hiroshi Miyano; Masayuki Sugiki

doi:10.1016/j.enzmictec.2023.110287

Development of a novel single-chain l-glutamate oxidase from Streptomyces sp. X-119-6 by inserting flexible linkers

Enzyme Microb Technol. 2023 Oct:170:110287. doi: 10.1016/j.enzmictec.2023.110287. Epub 2023 Jul 15.

Authors

Hiroki Yamaguchi¹, Kazutoshi Takahashi², Moemi Tatsumi², Uno Tagami², Toshimi Mizukoshi², Hiroshi Miyano², Masayuki Sugiki³

Affiliations

¹ Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan. Electronic address: hiroki.yamaguchi.wk7@asv.ajinomoto.com.
² Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan.
³ Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan. Electronic address: masayuki.sugiki.j56@asv.ajinomoto.com.

PMID: 37487431
DOI: 10.1016/j.enzmictec.2023.110287

Abstract

L-glutamate oxidase (LGOX, EC: 1.4.3.11) is an oxidoreductase that catalyzes L-glutamate deamination. LGOX from Streptomyces sp. X-119-6 is used widely for L-glutamate quantification in research and industrial applications. This enzyme encoded as a single precursor chain that undergoes post-translational cleavage to four fragments by an endogenous protease to become highly active. Efficient preparation of active LGOX by heterologous expression without proteolysis process should be indispensable for wide application of this enzyme. Thus, developing an LGOX that requires no protease treatment should expand the potential applications of recombinant LGOX. In this report, we succeeded in obtaining an active single-chain LGOX by connecting the four fragments of the mature form with insertion of flexible linkers. The most active single-chain mutant showed the similar activity to that of the mature form from Streptomyces sp. X-119-6. The structure of this mutant was determined at 2.9 Å resolution by X-ray crystallography. It was revealed that this single-stranded mutant had the similar conformation to that of mature form. This single-chain LGOX can be produced efficiently and should expand LGOX applications.

Keywords: Amino acids analysis; Enzymatic analysis; Enzyme engineering; Glutamate oxidase; Oxidoreductase; X-ray crystallography.