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Mol. Cells

Published online June 14, 2022

© The Korean Society for Molecular and Cellular Biology

The Crystal Structure of L-Leucine Dehydrogenase from Pseudomonas aeruginosa

Seheon Kim1 , Seri Koh1 , Wonchull Kang1,2,* , and Jin Kuk Yang1,*

1Department of Chemistry, College of Natural Sciences, Soongsil University, Seoul 06978, Korea, 2Department of Physics and Integrative Institute of Basic Science, Soongsil University, Seoul 06978, Korea

Correspondence to : jinkukyang@ssu.ac.kr(JKY); wonchullkang@ssu.ac.kr(WK)

Received: October 29, 2021; Revised: February 4, 2022; Accepted: February 24, 2022

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.

Abstract

Leucine dehydrogenase (LDH, EC 1.4.1.9) catalyzes the reversible deamination of branched-chain L-amino acids to their corresponding keto acids using NAD+ as a cofactor. LDH generally adopts an octameric structure with D4 symmetry, generating a molecular mass of approximately 400 kDa. Here, the crystal structure of the LDH from Pseudomonas aeruginosa (Pa-LDH) was determined at 2.5 Å resolution. Interestingly, the crystal structure shows that the enzyme exists as a dimer with C2 symmetry in a crystal lattice. The dimeric structure was also observed in solution using multiangle light scattering coupled with size-exclusion chromatography. The enzyme assay revealed that the specific activity was maximal at 60°C and pH 8.5. The kinetic parameters for three different amino acid and the cofactor (NAD+) were determined. The crystal structure represents that the subunit has more compact structure than homologs’ structure. In addition, the crystal structure along with sequence alignments indicates a set of non-conserved arginine residues which are important in stability. Subsequent mutation analysis for those residues revealed that the enzyme activity reduced to one third of the wild type. These results provide structural and biochemical insights for its future studies on its application for industrial purposes.

Keywords branched-chain amino acid, leucine dehydro­genase, Pseudomonas aeruginosa, PA3418

Article

On-line First

Mol. Cells

Published online June 14, 2022

Copyright © The Korean Society for Molecular and Cellular Biology.

The Crystal Structure of L-Leucine Dehydrogenase from Pseudomonas aeruginosa

Seheon Kim1 , Seri Koh1 , Wonchull Kang1,2,* , and Jin Kuk Yang1,*

1Department of Chemistry, College of Natural Sciences, Soongsil University, Seoul 06978, Korea, 2Department of Physics and Integrative Institute of Basic Science, Soongsil University, Seoul 06978, Korea

Correspondence to:jinkukyang@ssu.ac.kr(JKY); wonchullkang@ssu.ac.kr(WK)

Received: October 29, 2021; Revised: February 4, 2022; Accepted: February 24, 2022

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.

Abstract

Leucine dehydrogenase (LDH, EC 1.4.1.9) catalyzes the reversible deamination of branched-chain L-amino acids to their corresponding keto acids using NAD+ as a cofactor. LDH generally adopts an octameric structure with D4 symmetry, generating a molecular mass of approximately 400 kDa. Here, the crystal structure of the LDH from Pseudomonas aeruginosa (Pa-LDH) was determined at 2.5 Å resolution. Interestingly, the crystal structure shows that the enzyme exists as a dimer with C2 symmetry in a crystal lattice. The dimeric structure was also observed in solution using multiangle light scattering coupled with size-exclusion chromatography. The enzyme assay revealed that the specific activity was maximal at 60°C and pH 8.5. The kinetic parameters for three different amino acid and the cofactor (NAD+) were determined. The crystal structure represents that the subunit has more compact structure than homologs’ structure. In addition, the crystal structure along with sequence alignments indicates a set of non-conserved arginine residues which are important in stability. Subsequent mutation analysis for those residues revealed that the enzyme activity reduced to one third of the wild type. These results provide structural and biochemical insights for its future studies on its application for industrial purposes.

Keywords: branched-chain amino acid, leucine dehydro­genase, Pseudomonas aeruginosa, PA3418

Mol. Cells
Jun 30, 2022 Vol.45 No.6, pp. 353~434
COVER PICTURE
ERα is modified by UFM1 and this modification (ufmylation) plays a crucial role in promoting the stability of ERα and breast cancer development. However, when ERα is deufmylated and then ubiquitinated, it disappears by proteasome-mediated degradation (Yoo et al., pp. 425-434).

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