The full-length cytochrome P450 enzyme CYP102A1 dimerizes at its reductase domains and has flexible heme domains for efficient catalysis

Haoming Zhang,Adam L Yokom,Shen Cheng,Min Su,Paul F Hollenberg,Daniel R Southworth,Yoichi Osawa

doi:10.1074/jbc.ra117.000600

Haoming Zhang, Adam L Yokom + Show 5 more

Open Access

https://doi.org/10.1074/jbc.ra117.000600

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Abstract

The cytochrome P450 enzyme CYP102A1 from Bacillus megaterium is a highly efficient hydroxylase of fatty acids, and there is a significant interest in using CYP102A1 for biotechnological applications. Here, we used size-exclusion chromatography-multiangle light scattering (SEC-MALS) analysis and negative-stain EM to investigate the molecular architecture of CYP102A1. The SEC-MALS analysis yielded a homogeneous peak with an average molecular mass of 235 ± 5 kDa, consistent with homodimeric CYP102A1. The negative-stain EM of dimeric CYP102A1 revealed four distinct lobes, representing the two heme and two reductase domains. Two of the lobes were in close contact, whereas the other two were often observed apart and at the ends of a U-shaped configuration. The overall dimension of the dimer was ∼130 Å. To determine the identity of the lobes, we FLAG-tagged the N or C terminus of CYP102A1 to visualize additional densities in EM and found that anti-FLAG Fab could bind only the N-tagged P450. Single-particle analysis of this anti-Flag Fab-CYP102A1 complex revealed additional density in the N-terminally tagged heme domains, indicating that the heme domains appear flexible, whereas the reductase domains remain tightly associated. The effects of truncation on CYP102A1 dimerization, identification of cross-linked sites by peptide mapping, and molecular modeling results all were consistent with the dimerization of the reductase domain. We conclude that functional CYP102A1 is a compact globular protein dimerized at its reductase domains, with its heme domains exhibiting multiple conformations that likely contribute to the highly efficient catalysis of CYP102A1.

Highlights

The cytochrome P450 enzyme CYP102A1 from Bacillus megaterium is a highly efficient hydroxylase of fatty acids, and there is a significant interest in using CYP102A1 for biotechnological applications
We previously reported the architecture of neuronal nitric-oxide synthase (NOS) using negative stain EM. 2D analysis of reference free class averages showed flexible reductase domains around the oxygenase dimer interface [16]
The CYP102A1 elutes predominantly as a single peak exhibiting an average molecular mass value of 235 Ϯ 5 kDa (Fig. 1A). This indicates that CYP102A1 exists as a stable dimer in solution because the molecular mass is consistent with the theoretical mass of 238 kDa calculated from the amino acid sequence for a full-length CYP102A1 existing as a homodimer

Summary

ARTICLE cro

The negative-stain EM of dimeric CYP102A1 revealed four distinct lobes, representing the two heme and two reductase domains. Unlike other bacterial and mammalian CYP enzymes, the heme domain (BMP) and reductase domain (BMR) of CYP102A1 are fused in a single polypeptide chain with the N-terminal BMP domain being connected by a linker region to the C-terminal BMR domain This structural arrangement is analogous to nitric-oxide synthase (NOS) and believed to grant CYP102A1 extraordinary catalytic prowess for hydroxylating fatty acids. A single amino acid substitution of Ala Phe converts CYP102A1 from fatty acid hydroxylase to a human CYP2C19-like enzyme capable of oxidizing omeprazole at a rate of 1,460 minϪ1 [11] These mutagenic studies have provided a wealth of information regarding the roles of specific residues, largely in the catalytic BMP domain. Studies from Fab labeling, progressive truncation, chemical cross-linking, and molecular modeling are consistent with dimerization of the BMR domains with flexibility in the BMP domains

Results

Dimer kDa

Discussion

Experimental procedures