Aminobacter aminovorans NADH : Flavin oxidoreductase His140: A highly conserved residue critical for NADH binding and utilization

作者：Russell TR; Tu SC^*

来源：Biochemistry, 2004, 43(40): 12887-12893.

DOI：10.1021/bi048499n

Homodimeric FRDAa Class I is an NADH:flavin oxidoreductase from Aminobacter aminovorans. It is unusual because it contains an FMN cofactor but utilizes a sequential-ordered kinetic mechanism. Because little is known about NADH-specific flavin reductases in general and FRDAa in particular, this study aimed to further explore FRDAa by identifying the functionalities of a key residue. A sequence alignment of FRDAa with several known and hypothetical flavoproteins in the same subfamily reveals within the flavin reductase active-site domain a conserved GDH motif, which is believed to be responsible for the enzyme and NADH interaction. Mutation of the His 140 in this GDH motif to alanine reduced FRDAa activity to <3%. An ultrafiltration assay and fluorescence quenching demonstrated that H140A FRDAa binds FMN in the same 1:1 stoichiometric ratio as the wild-type enzyme, but with slightly weakened affinity (K-d = 0.9 muM). Anaerobic stopped-flow studies were carried out using both the native and mutated FRDAa. Similar to the native enzyme, H140A FRDAa was also able to reduce the FMN cofactor by NADH although much less efficiently. Kinetic analysis of anaerobic reduction measurements indicated that the His140 residue of FRDAa was essential to NADH binding, as well as important for the reduction of the FMN cofactor. For the native enzyme, the cofactor reduction was followed by at least one slower step in the catalytic pathway.

• 出版日期2004-10-12