Expression, Purification, and Characterization of Bacillus subtilis Cytochromes P450 CYP102A2 and CYP102A3:  Flavocytochrome Homologues of P450 BM3 from Bacillus megaterium

Abstract

The cyp102A2 and cyp102A3 genes encoding the two Bacillus subtilis homologues (CYP102A2 and CYP102A3) of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium have been cloned, expressed in Escherichia coli, purified, and characterized spectroscopically and enzymologically. Both enzymes contain heme, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) cofactors and bind a variety of fatty acid molecules, as demonstrated by conversion of the low-spin resting form of the heme iron to the high-spin form induced by substrate-binding. CYP102A2 and CYP102A3 catalyze the fatty acid-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduction of artificial electron acceptors at high rates. Binding of carbon monoxide to the reduced forms of both enzymes results in the shift of the heme Soret band to 450 nm, confirming the P450 nature of the enzymes. Reverse-phase high-performance liquid chromatography (HPLC) of products from the reaction of the enzymes with myristic acid demonstrates that both catalyze the subterminal hydroxylation of this substrate, though with different regioselectivity and catalytic rate. Both P450s 102A2 and 102A3 show kinetic and binding preferences for long-chain unsaturated and branched-chain fatty acids over saturated fatty acids, indicating that the former two molecule types may be the true substrates. P450s 102A2 and 102A3 exhibit differing substrate selectivity profiles from each other and from P450 BM3, indicating that they may fulfill subtly different cellular roles. Titration curves for binding and turnover kinetics of several fatty acid substrates with P450s 102A2 and 102A3 are better described by sigmoidal (rather than hyperbolic) functions, suggesting binding of more than one molecule of substrate to the P450s, or possibly cooperativity in substrate binding. Comparison of the amino acid sequences of the three flavocytochromes shows that several important amino acids in P450 BM3 are not conserved in the B. subtilis homologues, pointing to differences in the binding modes for the substrates that may explain the unusual sigmoidal kinetic and titration properties.

Unusual Cytochrome P450 Enzymes and Reactions

“…see Ref. 73). The physiological function of P450 BM-3 still remains unclear, although a role in bacterial quorum sensing mediated by oxidative inactivation of acylhomoserine lactones has been proposed (74).…”

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

“…Interestingly, whereas ω-C=C bonds increased the regioselectivity towards ω-2, probably due to the activated allylic location of hydroxylation (Table 6, entries 2, 3, and 9), a terminal alkyne-group was demonstrated to deactivate the enzyme by forming an adduct with the heme (Table 6, entry 10) [102,106]. The self-sufficiency of BM3 and its various reactions inspired the characterization of numerous additional CYP102 members, such as CYP102D1 from Streptomyces avermitiliswhich is the only characterized CYP102 outside the Bacillus family [107], CYP102A7 from Bacillus licheniformis (Table 5, entries 11, 30, and 40, Table 6, entries 8 and 13 and Table 7, entries 5 and 10) [78], and CYP102A2 and CYP102A3, both from Bacillus subtilis [108]. Similar as BM3, these family members prefer unsaturated or branched over saturated fatty acids and catalyze the in-chain hydroxylation of all three positions (ω-1, ω-2, and ω-3, e.g.…”

Chain length-dependent cooperativity in fatty acid binding and oxidation by cytochrome P450BM3 (CYP102A1)

“…The substrate binding and turnover kinetics of both A2 and A3 show sigmoidal behavior, indicating the binding of more than one substrate molecule and possible cooperativity. No sigmoidal behavior has been reported for fatty acid binding or oxidation by P450 BM3 (Gustafsson et al, 2004), though isotope effect studies suggested that both laurate and palmitate could be simultaneously present in the active site (Rock et al, 2003). On the other hand, both homotropic and heterotropic cooperativity have been reported for the oxidation of non-natural substrates such as indole by P450 BM3 (Li et al, 2005;Maurer et al, 2005;Huang et al, 2007), and for drug metabolism by the R47L/ F87V/L188Q mutant in which allosteric effects were also proposed (van Vugt-Lussenburg et al, 2006).…”

“…Of the A-subfamily enzymes only CYP102A1, A2, A3, A5 and A7 have been studied in detail. The A2 and A3 enzymes show a preference for branched-chain over linear-chain fatty acids (Budde et al, 2004;Gustafsson et al, 2004;Lentz et al, 2004), A5 prefers polyunsaturated fatty acids over their saturated counterparts , while A7 is broadly similar to the A1 enzyme (Dietrich et al, 2008). The substrate binding and turnover kinetics of both A2 and A3 show sigmoidal behavior, indicating the binding of more than one substrate molecule and possible cooperativity.…”