Polyol-specific long-chain dehydrogenases/reductases of mannitol metabolism in Aspergillus fumigatus: Biochemical characterization and pH studies of mannitol 2-dehydrogenase and mannitol-1-phosphate 5-dehydrogenase

Abstract

Functional genomics data suggests that the metabolism of mannitol in the human pathogen Aspergillus fumigatus involves the action of two polyol-specific long-chain dehydrogenases/reductases, mannitol-1-phosphate 5-dehydrogenase (M1PDH) and mannitol 2-dehydrogenase (M2DH). The gene encoding the putative M2DH was expressed in Escherichia coli, and the purified recombinant protein was characterized biochemically. The predicted enzymatic function of a NAD +-dependent M2DH was confirmed. The enzyme is a monomer of 58 kDa in solution and does not require metals for activity. pH profiles for M2DH and the previously isolated M1PDH were recorded in the pH range 6.0–10.0 for the oxidative and reductive direction of the reactions under conditions where substrate was limiting ( k cat/ K) or saturating ( k cat). The pH-dependence of log k cat was usually different from that of log( k cat/ K), suggesting that more than one step of the enzymatic mechanism was affected by changes in pH. The greater complexity of the pH profiles of log( k cat/ K) for the fungal enzymes as compared to the analogous pH profiles for M2DH from Pseudomonas fluorescens may reflect sequence changes in vicinity of the conserved catalytic lysine.