Kinetic Mechanism and Quaternary Structure of Aminobacter aminovorans NADH:Flavin Oxidoreductase: An Unusual Flavin Reductase with Bound Flavin
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- 作者：Thomas R. Russell ; Borries Demeler ; and Shiao-Chun Tu
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：February 17, 2004
- 年：2004
- 卷：43
- 期：6
- 页码：1580 - 1590
- 全文大小：195K
- 年卷期：v.43,no.6(February 17, 2004)
- ISSN：1520-4995
文摘
The homodimeric NADH:flavin oxidoreductase from Aminobacter aminovorans is an NADH-specific flavin reductase herein designated FRDAa. FRDAa was characterized with respect to purificationyields, thermal stability, isoelectric point, molar absorption coefficient, and effects of phosphate bufferstrength and pH on activity. Evidence from this work favors the classification of FRDAa as a flavin cofactor-utilizing class I flavin reductase. The isolated native FRDAa contained about 0.5 bound riboflavin-5'-phosphate (FMN) per enzyme monomer, but one bound flavin cofactor per monomer was obtainable inthe presence of excess FMN or riboflavin. In addition, FRDAa holoenzyme also utilized FMN, riboflavin,or FAD as a substrate. Steady-state kinetic results of substrate titrations, dead-end inhibition by AMP andlumichrome, and product inhibition by NAD+ indicated an ordered sequential mechanism with NADH asthe first binding substrate and reduced FMN as the first leaving product. This is contrary to the ping-pongmechanism shown by other class I flavin reductases. The FMN bound to the native FRDAa can be fullyreduced by NADH and subsequently reoxidized by oxygen. No NADH binding was detected using 90
M FRDAa apoenzyme and 300 M NADH. All results favor the interpretation that the bound FMN wasa cofactor rather than a substrate. It is highly unusual that a flavin reductase using a sequential mechanismwould require a flavin cofactor to facilitate redox exchange between NADH and a flavin substrate. FRDAaexhibited a monomer-dimer equilibrium with a Kd of 2.7 M. Similarities and differences between FRDAaand certain flavin reductases are discussed.
M FRDAa apoenzyme and 300 M NADH. All results favor the interpretation that the bound FMN wasa cofactor rather than a substrate. It is highly unusual that a flavin reductase using a sequential mechanismwould require a flavin cofactor to facilitate redox exchange between NADH and a flavin substrate. FRDAaexhibited a monomer-dimer equilibrium with a Kd of 2.7 M. Similarities and differences between FRDAaand certain flavin reductases are discussed.