Dihydrolipoamide dehydrogenase from Leishmania donovani: New insights through biochemical characterization.

Dihydrolipoamide dehydrogenase (DLDH) regulates many crucial metabolic pathways as a multi-enzyme complex. Leishmania donovani dihydrolipoamide dehydrogenase (LdDLDH) has two variants present on two different chromosomes with very less sequence similarities. In the current study, we cloned both the variants in pET28a (+) vector and expressed in Rosetta-gami (DE3) E. coli strain. Expressed proteins were finally purified from pellets using Ni-NTA affinity chromatography. Purified enzymes were biochemically characterized and different kinetic parameters were studied. Both the variants showed maximum activity in pH range of 7.0-8.0 and temperature 50±5°C in the physiological direction. The estimated K for dihydrolipoamide (DLA) and NAD were 2.7±0.48mM and 171.23±11.59μM respectively for variant 1 (LdBPK291950.1). In the case of variant 2 (LdBPK323510.1), K values for DLA and NAD were found to be 829.85±37μM and 226±1.56μM respectively. The variant 2 was more efficient in terms of activity. While both the forms of the enzymes showed diaphorase activity, variant 1 was found to be better. Sequence dissimilarities of both forms were analyzed for biological insights.