Crystal Structure of H2O2-dependent Cytochrome P450SPα with Its Bound Fatty Acid Substrate

Abstract

Cytochrome P450 SP␣ (CYP152B1) isolated from Sphingomonas paucimobilis is the first P450 to be classified as a H 2 O 2 -dependent P450. P450 SP␣ hydroxylates fatty acids with high ␣-regioselectivity. Herein we report the crystal structure of P450 SP␣ with palmitic acid as a substrate at a resolution of 1.65 Å . The structure revealed that the C ␣ of the bound palmitic acid in one of the alternative conformations is 4.5 Å from the heme iron. This conformation explains the highly selective ␣-hydroxylation of fatty acid observed in P450 SP␣ . Mutations at the active site and the F-G loop of P450 SP␣ did not impair its regioselectivity. The crystal structures of mutants (L78F and F288G) revealed that the location of the bound palmitic acid was essentially the same as that in the WT, although amino acids at the active site were replaced with the corresponding amino acids of cytochrome P450 BS␤ (CYP152A1), which shows ␤-regioselectivity. This implies that the high regioselectivity of P450 SP␣ is caused by the orientation of the hydrophobic channel, which is more perpendicular to the heme plane than that of P450 BS␤ .

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

“…111 The opposite is true for mutations to the P450 BSb active site, which are capable of switching the regiochemistry of oxidation by this P450 towards the a-position. 111 The use of hydrogen peroxide as an oxidant means that there is not the need to interact with redox partner proteins, and this is reected in an extended loop prior to the axial cysteine ligand on the proximal face of the heme coupled with a predominant negative charge, which differs to P450s that interact with redox partner proteins. 111,112 The use of peroxide by these P450s accounts for their rapid rate of turnover and also serves to explain the high levels of interest in such enzymes as potential biocatalysts, although inactivation by high levels of peroxide can limit the usefulness of this approach.…”

“…[111][112][113] The regioselectivity of P450 SPa appears to be maintained through the channel that orients the fatty acid within the active site, as mutations corresponding the P450 BSb active site are unable to switch regioselectivity towards the b-position. 111 The opposite is true for mutations to the P450 BSb active site, which are capable of switching the regiochemistry of oxidation by this P450 towards the a-position. 111 The use of hydrogen peroxide as an oxidant means that there is not the need to interact with redox partner proteins, and this is reected in an extended loop prior to the axial cysteine ligand on the proximal face of the heme coupled with a predominant negative charge, which differs to P450s that interact with redox partner proteins.…”

“…111 The use of hydrogen peroxide as an oxidant means that there is not the need to interact with redox partner proteins, and this is reected in an extended loop prior to the axial cysteine ligand on the proximal face of the heme coupled with a predominant negative charge, which differs to P450s that interact with redox partner proteins. 111,112 The use of peroxide by these P450s accounts for their rapid rate of turnover and also serves to explain the high levels of interest in such enzymes as potential biocatalysts, although inactivation by high levels of peroxide can limit the usefulness of this approach. 114 An alternate solution has been demonstrated in the case of the related a-hydroxylase CYP152A2 from Clostridium acetobutylicum, in which reconstitution with a functional redox partner chain was able to improve the catalytic properties of this enzyme.…”

“…Specic arginine (R241/R242) residues in P450 SPa and P450 BSb are crucial as they bind the carboxylate of the fatty acid to position the substrate above the heme for oxidation in both cases. 111,112 Moreover, the carboxylic acid moiety appears to play a role in the generation of compound I from hydrogen peroxide in these P450s, which is an important example of substrateassistance in the P450 active cycle. [111][112][113] The regioselectivity of P450 SPa appears to be maintained through the channel that orients the fatty acid within the active site, as mutations corresponding the P450 BSb active site are unable to switch regioselectivity towards the b-position.…”

“…111,112 Moreover, the carboxylic acid moiety appears to play a role in the generation of compound I from hydrogen peroxide in these P450s, which is an important example of substrateassistance in the P450 active cycle. [111][112][113] The regioselectivity of P450 SPa appears to be maintained through the channel that orients the fatty acid within the active site, as mutations corresponding the P450 BSb active site are unable to switch regioselectivity towards the b-position. 111 The opposite is true for mutations to the P450 BSb active site, which are capable of switching the regiochemistry of oxidation by this P450 towards the a-position.…”