.2025 Jan 6;177(1):27-36.

doi: 10.1093/jb/mvae070.

Open and closed structures of L-arginine oxidase by cryo-electron microscopy and X-ray crystallography

Hiroki Yamaguchi^{1 2}, Kazutoshi Takahashi¹, Nobutaka Numoto³, Hiroshi Suzuki², Moemi Tatsumi¹, Akiko Kamegawa^{2 4 5}, Kouki Nishikawa^{4 5}, Yasuhisa Asano⁶, Toshimi Mizukoshi¹, Hiroshi Miyano¹, Yoshinori Fujiyoshi^{2 4 5}, Masayuki Sugiki¹

Affiliations

¹ Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co. Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan.
² Advanced Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8501, Japan.
³ Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8501, Japan.
⁴ CeSPIA Inc., 2-1-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan.
⁵ Joint Research Course for Advanced Biomolecular Characterization, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
⁶ Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.

PMID:39420599
PMCID: PMC11694665
DOI: 10.1093/jb/mvae070

Open and closed structures of L-arginine oxidase by cryo-electron microscopy and X-ray crystallography

Hiroki Yamaguchi et al. J Biochem.2025.

.2025 Jan 6;177(1):27-36.

doi: 10.1093/jb/mvae070.

Authors

Affiliations

¹ Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co. Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan.
² Advanced Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8501, Japan.
³ Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8501, Japan.
⁴ CeSPIA Inc., 2-1-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan.
⁵ Joint Research Course for Advanced Biomolecular Characterization, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
⁶ Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan.

PMID:39420599
PMCID: PMC11694665
DOI: 10.1093/jb/mvae070

Abstract

L-arginine oxidase (AROD, EC 1.4.3.25) is an oxidoreductase that catalyses the deamination of L-arginine, with flavin adenine dinucleotide (FAD) as a cofactor. Recently identified AROD from Pseudomonas sp. TPU 7192 (PT-AROD) demonstrates high selectivity for L-arginine. This enzyme is useful for accurate assays of L-arginine in biological samples. The structural characteristics of the FAD-dependent AROD, however, remain unknown. Here, we report the structure of PT-AROD at a resolution of 2.3 Å by cryo-electron microscopy. PT-AROD adopts an octameric structure with D4 symmetry, which is consistent with its molecular weight in solution, estimated by mass photometry. Comparative analysis of this structure with that determined using X-ray crystallography reveals open and closed forms of the lid-like loop at the entrance to the substrate pocket. Furthermore, mutation of Glu493, located at the substrate binding site, diminishes substrate selectivity, suggesting that this residue contributes significantly to the high selectivity of PT-AROD.

Keywords: L-arginine oxidase; X-ray crystallography; amino acid oxidase; cryo-EM; single particle analysis.

PubMed Disclaimer

Figures

**Fig. 1**
**Cryo-EM analysis flow chart of the cryo-EM data processing for L-arginine oxidase fromPseudomonas sp. TPU 7192.** Processing workflow for PT-AROD structure determination by single-particle analysis. Gold-standard FSC curves (right bottom) for PT-AROD are displayed after applying no mask (green), a mask (blue) or phase-randomized mask (red). The corrected FSC curve is shown in black. All images in this figure were created in UCSF Chimera.

**Fig. 2**
**Cryo-EM map of L-arginine oxidase fromPseudomonas sp. TPU 7192.** The density map of PT-AROD is shown at a contour level 0.05. Density maps applicable to each monomer are shown in different colours. All images in this figure were created in UCSF ChimeraX.

**Fig. 3**
**Structural comparison between Cryo-EM and X-ray crystallography.** (A) PT-AROD A chain (cyan) and B–H chain (white) from cryo-EM are shown as a cartoon model. Three residues (G213–E215) are disordered, and the ATEYS (Ala-Thr-Glu-Tyr-Ser) loop (A222–S226) differs. (B) Superimposed cryo-EM (cyan) and X-ray crystallography (white) structures. Dotted circle shows the ATEYS loop. (C) Side view of the substrate binding pocket. The cryo-EM structure is in an open conformation (left), whilst the X-ray crystallography structure is in a closed conformation (right). PT-AROD is shown in surface representation, and FAD is shown as a stick model. (D) The cryo-EM (left) and X-ray crystallography (right) structures of PT-AROD are coloured according to the temperature factor. Black arrows indicate the ATEYS loops.

**Fig. 4**
**Substrate binding site of L-arginine oxidase and L-lysine oxidase.** (Left) Superposition of FAD-bound monomeric subunits of PT-AROD (cyan, cryo-EM) and AncLLysO (magenta, PDB: 7EIH). (Middle) Close-up view of the residues around the putative substrate site, shown as a stick model. (Right) Comparison of the substrate sites of PT-AROD/FAD (cyan, cryo-EM), AncLLysO/FAD/L-arginine complexes (yellow, PDB: 7EIJ) and AncLLysO/FAD/L-lysine (orange, PDB: 7EII). Dotted line indicates a hydrogen bond between Glu383 and L-arginine side chains.

See this image and copyright information in PMC

References

1. Yoshida, H., Kondo, K., Yamamoto, H., Kageyama, N., Ozawa, S., Shimbo, K., Muramatsu, T., Imaizumi, A., Mizukoshi, T., Masuda, J., Nakayama, D., Hayakawa, Y., Watanabe, K., Mukaibatake, K., and Miyano, H. (2015) Validation of an analytical method for human plasma free amino acids by high-performance liquid chromatography ionization mass spectrometry using automated precolumn derivatization. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 998-999, 88–96 - PubMed
1. Shimbo, K., Oonuki, T., Yahashi, A., Hirayama, K., and Miyano, H. (2009) Precolumn derivatization reagents for high-speed analysis of amines and amino acids in biological fluid using liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun. Mass Spectrom. 23, 1483–1492 - PubMed
1. Spackman, D.H., Stein, W.H., and Moore, S. (1958) Automatic recording apparatus for use in chromatography of amino acids. Anal. Chem. 30, 1190–1206 - PubMed
1. Matsuda, M. and Asano, Y. (2010) Determination of plasma and serum L-lysine using L-lysine epsilon-oxidase from Marinomonas mediterranea NBRC 103028(T). Anal. Biochem. 406, 19–23 - PubMed
1. Kameya, M., Onaka, H., and Asano, Y. (2013) Selective tryptophan determination using tryptophan oxidases involved in bis-indole antibiotic biosynthesis. Anal. Biochem. 438, 124–132 - PubMed

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions

Movatterモバイル変換

Account

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Full text links

Actions

Share

Open and closed structures of L-arginine oxidase by cryo-electron microscopy and X-ray crystallography

Affiliations

Open and closed structures of L-arginine oxidase by cryo-electron microscopy and X-ray crystallography

Authors

Affiliations

Abstract

Figures

Similar articles

References

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources