PMID: 8463285Apr 5, 1993Paper

Critical residues involved in FMN binding and catalytic activity in cytochrome P450BM-3.

The Journal of Biological Chemistry
M L Klein, A J Fulco

Abstract

Cytochrome P450BM-3 from Bacillus megaterium is a soluble, catalytically self-sufficient fatty acid mono-oxygenase that, in structural organization and amino acid sequence, resembles the Class II (microsomal) P450 systems. Its single polypeptide chain contains both a P450 heme domain and an NADPH:P450 reductase domain, each of which bears significant homology with its microsomal counterparts. We report here the critical nature of three amino acids in the reductase domain of this enzyme with respect to FMN binding and catalytic activity. We used site-directed mutagenesis to change glycine 570 to bulkier amino acids; none of these mutant enzymes contained FMN after purification. We also made substitutions for tryptophan 574 and tyrosine 536, which by sequence analogy (Porter, T. D. (1991) Trends Biochem. Sci. 16, 154-158) were proposed to bind FMN through stacking of the aromatic rings with the isoalloxazine ring of the flavin. Mutants of tryptophan 574 which retained the aromatic side chain contained no less than 0.85 mol of FMN per mol of enzyme, while aspartate and glycine substitutions yielded enzymes which did not incorporate FMN. Substitution of tyrosine 536 with aspartate gave an enzyme which contained 0.44 mol of FMN per ...Continue Reading

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