Mutational studies on Leishmania donovani dihydrolipoamide dehydrogenase (LdBPK291950.1) indicates that the enzyme may not be classical class-I pyridine nucleotide-disulfide oxidoreductase

Int J Biol Macromol. 2020 Dec 1:164:2141-2150. doi: 10.1016/j.ijbiomac.2020.07.236. Epub 2020 Aug 1.

Abstract

We report biochemical studies on two Cys residues mutation (Cys15Thr, Cys38Gly) nearest to the active site and three other amino acid substitution mutations expected to be the part of active site of LdDLDH_Variant1. Our biochemical studies show that the replacement of Cys15 increases the Km for dihydrolipoamide (DLD) substrate by five folds and NAD+ by three fold indicating that this mutation affects the binding of DLD and NAD+ significantly. Cys38 was also mutated to 'Gly' which resulted in nine fold greater Km for NAD+ without affecting Km for DLD. However, even after these mutations (Cys15Thr and Cys38Gly), reduced enzyme activity suggests that both the 'Cys' residues are not involved in disulfide bond formation but affect the binding of substrates. The data hints towards the possibility of a different catalytic mechanism from the classical class I - pyridine nucleotide-disulfide oxidoreductase. Remaining other mutated residues Ala48Ile, Asp49Gly, and Ala54Ile showed an increase in two to three-folds Km value for NAD+, which means these residues are important for the binding of NAD+ to the enzyme. However, Ala48Ile and Asp49Gly mutations showed a decrease of Km for DLD. Apart from the mutational studies, localization of LdDLDH_Variant2 of LdDLDH was also analyzed.

Keywords: Dihydrolipoamide dehydrogenase; Leishmania; Mutation; Oxidoreductase.

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution / genetics
  • Catalysis
  • Catalytic Domain / genetics
  • Dihydrolipoamide Dehydrogenase / genetics*
  • Disulfides / metabolism*
  • Leishmania donovani / enzymology*
  • Leishmania donovani / genetics*
  • Mutation / genetics*
  • NAD / genetics
  • Nucleotides / genetics*
  • Oxidoreductases / genetics*
  • Oxidoreductases / metabolism
  • Pyridines / metabolism*
  • Sequence Alignment / methods
  • Thioctic Acid / analogs & derivatives
  • Thioctic Acid / genetics

Substances

  • Disulfides
  • Nucleotides
  • Pyridines
  • NAD
  • dihydrolipoamide
  • Thioctic Acid
  • Oxidoreductases
  • Dihydrolipoamide Dehydrogenase