Active Site Substitution A82W Improves the Regioselectivity of Steroid Hydroxylation by Cytochrome P450 BM3 Mutants As Rationalized by Spin Relaxation Nuclear Magnetic Resonance Studies

作者：Rea V; Kolkman A J; Vottero E; Stronks E J; Ampt K A M; Honing M; Vermeulen N P E; Wijmenga S S; Commandeur J N M^*

来源：Biochemistry, 2012, 51(3): 750-760.

DOI：10.1021/bi201433h

Cytochrome P450 BM3 from Bacillus megaterium is a monooxygenase with great potential for biotechnological applications. In this paper, we present engineered drug-metabolizing P450 BM3 mutants as a novel tool for regioselective hydroxylation of steroids at position 16 beta. In particular, we show that by replacing alanine at position 82 with a tryptophan in P450 BM3 mutants M01 and M11, the selectivity toward 16 beta-hydroxylation for both testosterone and norethisterone was strongly increased. The A82W mutation led to a %26lt;= 42-fold increase in V-max for 16 beta-hydroxylation of these steroids. Moreover, this mutation improves the coupling efficiency of the enzyme, which might be explained by a more efficient exclusion of water from the active site. The substrate affinity for testosterone increased at least 9-fold in M11 with tryptophan at position 82. A change in the orientation of testosterone in the M11 A82W mutant as compared to the orientation in M11 was observed by T-1 paramagnetic relaxation nuclear magnetic resonance. Testosterone is oriented in M11 with both the A- and D-ring protons closest to the heme iron. Substituting alanine at position 82 with tryptophan results in increased A-ring proton-iron distances, consistent with the relative decrease in the level of A-ring hydroxylation at position 2 beta.

• 出版日期2012-1-24