2001
DOI: 10.1021/bi011197q
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Pivotal Role of Water in the Mechanism of P450BM-3

Abstract: Cytochrome P450s constitute a superfamily of enzymes that catalyze the oxidation of a vast number of structurally and chemically diverse hydrophobic substrates. Herein, we describe the crystal structure of a complex between the bacterial P450BM-3 and the novel substrate N-palmitoylglycine at a resolution of 1.65 A, which reveals previously unrecognizable features of active site reorganization upon substrate binding. N-palmitoylglycine binds with higher affinity than any other known substrate and reacts with a … Show more

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Cited by 327 publications
(492 citation statements)
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“…The mechanism by which four palmitate molecules could bind to each monomer of dimeric full-length P450 BM3 or the heme domain is not clear. Crystal structures of the heme domain complexed with a fatty acid or derivative show only one substrate molecule in the active site, and in all cases the heme iron is penta-coordinate and almost certainly high spin (Hasemann et al, 1995;Li and Poulos, 1997;Haines et al, 2001;Joyce et al, 2004;Hegde et al, 2007;Huang et al, 2007;Haines et al, 2008). However, limited fatty acid solubility, the high protein concentrations and ionic strengths required for crystallization, and crystal packing effects mean that multiple fatty acid binding may not be observable in crystal structures.…”
Section: Discussionmentioning
confidence: 99%
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“…The mechanism by which four palmitate molecules could bind to each monomer of dimeric full-length P450 BM3 or the heme domain is not clear. Crystal structures of the heme domain complexed with a fatty acid or derivative show only one substrate molecule in the active site, and in all cases the heme iron is penta-coordinate and almost certainly high spin (Hasemann et al, 1995;Li and Poulos, 1997;Haines et al, 2001;Joyce et al, 2004;Hegde et al, 2007;Huang et al, 2007;Haines et al, 2008). However, limited fatty acid solubility, the high protein concentrations and ionic strengths required for crystallization, and crystal packing effects mean that multiple fatty acid binding may not be observable in crystal structures.…”
Section: Discussionmentioning
confidence: 99%
“…movement of the iron that directly impacts on the iron-ligand interactions, or movement of the palmitate molecule within the active site that enables a water molecule to ligate to, or dissociate from, the heme iron. A hydrophobic patch close to the mouth of the substrate access channel has been speculated to be the initial recognition site for fatty acids before the aliphatic chain enters the channel (Hasemann et al, 1995;Li and Poulos, 1997;Haines et al, 2001). If this patch remains accessible after the first fatty acid is bound, it may be an allosteric binding site.…”
Section: Discussionmentioning
confidence: 99%
“…Data were processed using XDS [27] and scaled using AIMLESS [28]. The structure was solved by molecular replacement using Molrep [29] from the CCP4 suite [30] and the coordinates of the heme domain of P450 BM3 structure (PDB 1JPZ) [22]. The models were adjusted using Coot [31] and refined using Refmac5 [32] and PHENIX [33].…”
Section: Methodsmentioning
confidence: 99%
“…The crystal structure of A2 mutant in complex with N-palmytoilglycine (NPG), a known substrate of P450 BM3 [22], was also solved and the electron density of the substrate was visible in one of the monomer ( Figure 4A). The overall structure is very similar to that of substrate-free A2 mutant (RMSD 0.583 Å).…”
Section: Crystal Structures Of the Heme Domain Of A2mentioning
confidence: 99%
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