Leucine dehydrogenase (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. Leucine dehydrogenase generally adopts an octameric structure with D4 symmetry, generating a molecular mass of approximately 400 kDa. Here, the crystal structure of the leucine dehydrogenase 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 is 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.
Leucine dehydrogenase(EC 1.4.1.9)는 NAD+ cofactor를 사용하여 branched-chain L-amino acid를 keto acid로 변환시키는 deamination 가역반응을 촉매한다. 이번 연구에서 Pseudomonas aeruginosa의 leucine dehydrogenase(Pa-LDH)의 구조를 2.5Å 해상도로 해석하였다. 흥미롭게도, Pa-LDH는 결정 격자 상태에서 C2 symmetry를 가진 dimer로 존재함을 보였다. 또한, dimeric 구조는 SEC coupled MALS 분석 결과에서도 관찰되었다. Enzyme assay에서는 60℃, pH8.5 조건에서 최대 specific activity를 보였다. 세 개의 substrates(L-leucine, L-isoleucine, L-valine)와 cofactor인 NAD+에 대한 kinetic parameter값을 얻었다. 결정 구조는 다른 homolog들의 구조보다 좀 더 compact한 구조를 갖는 것을 보였다. Sequence alignment를 통해 분석했을 때, Pa-LDH의 stability에 중요한 몇 가지 non-conserved arginine residue가 있음을 보였다. 이 residue들에 대한 mutation analysis 결과, wild type의 enzyme activity가 1/3로 줄어든 것을 확인하였다. 이러한 결과들은 산업 분야로의 적용을 위한 이후의 연구들에 대한 structural, biochemical insights을 제공한다.