1ZO9

Crystal Structure Of The Wild Type Heme Domain Of P450BM-3 with N-palmitoylmethionine

Summary for 1ZO9

• Entry DOI: 10.2210/pdb1zo9/pdb
• Related: 1BU7 1JPZ 1ZO4 1ZOA 2HPD
• Descriptor: Bifunctional P-450:NADPH-P450 reductase, PROTOPORPHYRIN IX CONTAINING FE, N-PALMITOYL-L-METHIONINE, ... (6 entities in total)
• Functional Keywords: cytochrome p-450, wild type heme protein, oxidoreductase
• Biological source: Bacillus megaterium
• Cellular location: Cytoplasm (By similarity): P14779
• Total number of polymer chains: 2
• Total formula weight: 111484.85

Authors

Hegda, A.,Chen, B.,Tomchick, D.R.,Bondlela, M.,Haines, D.C.,Schaffer, N.,Machius, M.,Graham, S.E.,Peterson, J.A. (deposition date: 2005-05-12, release date: 2006-08-01, Last modification date: 2023-08-23)

Primary citation

Hegde, A.,Haines, D.C.,Bondlela, M.,Chen, B.,Schaffer, N.,Tomchick, D.R.,Machius, M.,Nguyen, H.,Chowdhary, P.K.,Stewart, L.,Lopez, C.,Peterson, J.A.
Interactions of substrates at the surface of P450s can greatly enhance substrate potency.
Biochemistry, 46:14010-14017, 2007

PubMed Abstract: 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.

PubMed: 18004886
DOI: 10.1021/bi701667m

Experimental method

X-RAY DIFFRACTION (1.7 Å)