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.
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 58kDa 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 logk(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.
Krahulec
Stefan
S
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Graz, Austria.
Armao
Guilliano C
GC
Bubner
Patricia
P
Klimacek
Mario
M
Nidetzky
Bernd
B
D000409:Alanine / Q000235:genetics; D001232:Aspergillus fumigatus / Q000378:metabolism*; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000235:genetics; D008353:Mannitol / Q000378:metabolism*; D008354:Mannitol Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011108:Polymers / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D011108:Polymers; D011994:Recombinant Proteins; C024617:polyol; D008353:Mannitol; D008354:Mannitol Dehydrogenases; D013401:Sugar Alcohol Dehydrogenases; C021544:mannitol-1-phosphate dehydrogenase; D008239:Lysine; D000409:Alanine