Cytochrome P450s are a superfamily of heme containing enzymes that use molecular oxygen and electrons from reduced nicotinamide cofactors to monooxygenate organic substrates. The fatty acid hydroxylase P450BM-3 has been particularly widely studied due to its stability, high activity, similarity to mammalian P450s, and presence of a cytochrome P450 reductase domain that allows the enzyme to directly receive electrons from NADPH without a requirement for additional redox proteins. We previously characterized the substrate N-palmitoylglycine, which found extensive use in studies of P450BM-3 due to its high affinity, high turnover number, and increased solubility as compared to fatty acid substrates. Here, we report that even higher affinity substrates can be designed by acylation of other amino acids, resulting in P450BM-3 substrates with dissociation constants below 100 nM. N-Palmitoyl-l-leucine and N-palmitoyl-l-methionine were found to have the highest affinity, with dissociation constants of less than 8 nM and turnover numbers similar to palmitic acid and N-palmitoylglycine. The interactions of the amino acid side chains with a hydrophobic pocket near R47, as revealed by our crystal structure determination of N-palmitoyl-l-methionine bound to the heme domain of P450BM-3, appears to be responsible for increasing the affinity of substrates. The side chain of R47, previously shown to be important in interactions with negatively charged substrates, does not interact strongly with N-palmitoyl-l-methionine and is found positioned at the enzyme-solvent interface. These are the tightest binding substrates for P450BM-3 reported to date, and the affinity likely approaches the maximum attainable affinity for the binding of substrates of this size to P450BM-3.
P450BM-3 is an extensively studied P450 cytochrome that is naturally fused to a cytochrome P450 reductase domain. Crystal structures of the heme domain of this enzyme have previously generated many insights into features of P450 structure, substrate binding specificity, and conformational changes that occur on substrate binding. Although many P450s are inhibited by imidazole, this compound does not effectively inhibit P450BM-3. Omega-imidazolyl fatty acids have previously been found to be weak inhibitors of the enzyme and show some unusual cooperativity with the substrate lauric acid. We set out to improve the properties of these inhibitors by attaching the omega-imidazolyl fatty acid to the nitrogen of an amino acid group, a tactic that we used previously to increase the potency of substrates. The resulting inhibitors were significantly more potent than their parent compounds lacking the amino acid group. A crystal structure of one of the new inhibitors bound to the heme domain of P450BM-3 reveals that the mode of interaction of the amino acid group with the enzyme is different from that previously observed for acyl amino acid substrates. Further, required movements of residues in the active site to accommodate the imidazole group provide an explanation for the low affinity of imidazole itself. Finally, the previously observed cooperativity with lauric acid is explained by a surprisingly open substrate-access channel lined with hydrophobic residues that could potentially accommodate lauric acid in addition to the inhibitor itself.
Identifying key structural features of cytochromes P450 is critical in understanding the catalytic mechanism of these important drug-metabolizing enzymes. Cytochrome P450BM-3 (BM-3), a structural and mechanistic P450 model, catalyzes the regio- and stereoselective hydroxylation of fatty acids. Recent work has demonstrated the importance of water in the mechanism of BM-3, and site- specific mutagenesis has helped to elucidate mechanisms of substrate recognition, binding, and product formation. One of the amino acids identified as playing a key role in the active site of BM-3 is alanine 328, which is located in the loop between the K helix and β 1–4. In the A328V BM-3 mutant, substrate affinity increases 5 to 10-fold and the turnover number increases 2 to 8-fold compared to wild-type enzyme. Unlike wild-type enzyme, this mutant is purified from E. coli with endogenous substrate bound due to the higher binding affinity. Close examination of the crystal structures of the substrate-bound native and A328V mutant BMPs indicate that the positioning of the substrate is essentially identical in the two forms of the enzyme, with the two valine methyl groups occupying voids present in the active site of the wild-type substrate-bound structure.
The expression of lysozyme gene in the oviduct of Japanese quail is age-dependent. Here we show that the expression of the gene is altered by three steroid hormones: 17beta-estradiol (E), progesterone (P) and glucocorticoid (dexamethasone, G), and their combinations E+P, E+G and P+G. We also show that the levels/affinities of transacting factors that bind to specific cis-acting elements in the promoter region of the gene change with age and after steroid administration. These factors are sequence-specific, age and steroid-dependent. It is proposed that administration of appropriate doses of steroid hormones after adulthood may extend the reproductive function and egg laying period in birds.
Cytochrome P450 enzymes are important in Phase I xenobiotic metabolism, hormone metabolism, and the synthesis of numerous natural products. The bacterial enzyme P450BM‐3 has long served as a convenient model of P450 structure and function. As with many other P450s, P450BM‐3 undergoes a change in the spin‐state of the heme iron upon substrate binding. This change from the resting six‐coordinate low‐spin state to the five‐coordinate highspin state may help conserve electrons in the absence of oxidizable substrate, as the low‐spin state is harder to reduce. We have found that mutation of an active site alanine (A328) to valine produces an enzyme with greatly improved substrate binding, spin‐state conversion, and turnover rate. Mutation of the same residue to serine, however, results in greatly impaired spin‐state conversion and impaired substrate binding. Even more intriguing, the effect of the A328S mutant on Vmax depends on the type of substrate examine, with fatty acid oxidation being highly impaired but N‐acyl amino acid oxidation rate being enhanced ~60%. These results give us new insights into how protein conformational change couples substrate binding to spin‐state change. This research was supported in part by research grants GM43479 and GM50858 from the NIH (JAP) and X‐011 from the Robert A. Welch Foundation (DCH).
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