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Mol. Cells 2022; 45(7): 495-501

Published online July 31, 2022

https://doi.org/10.14348/molcells.2022.0012

© 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, PA3418, Pseudomonas aeruginosa

Article

Research Article

Mol. Cells 2022; 45(7): 495-501

Published online July 31, 2022 https://doi.org/10.14348/molcells.2022.0012

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, PA3418, Pseudomonas aeruginosa

Mol. Cells
Jul 31, 2022 Vol.45 No.7, pp. 435~512
COVER PICTURE
Mesenchymal stem cells (MSCs) are multipotent stem cells capable of differentiating into mesodermal lineages like adipogenic, osteogenic, and chondrogenic. Alcian blue-positive extracellular matrix secreted by chondrocytes in the lacuna confirmed the chondrogenic differentiation of MSCs (Bashyal et al., pp. 479-494).

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