Thr268 in Substrate Binding and Catalysis in P450BM-3

doi:10.1006/abbi.1997.0400

Archives of Biochemistry and Biophysics

Volume 349, Issue 1, 1 January 1998, Pages 53-64

https://doi.org/10.1006/abbi.1997.0400 Get rights and content

Abstract

Members of the gene superfamily of proteins called “P450” catalyze monooxygenation reactions that require an input of two electrons and a molecule of oxygen per catalytic cycle. These proteins are widely distributed among living organisms, from bacteria to human. P450BM-3, a soluble protein isolated fromBacillus megaterium,is self-sufficient, containing P450 and reductase domains on the same polypeptide. P450BM-3 catalyzes the hydroxylation of various fatty acids at ω-1, ω-2, and ω-3 positions, as well as epoxidations of double bonds. We have constructed the active-site mutant, T268A, and analyzed the effect on arachidonic acid and palmitic acid oxidation. Data indicate that the mutation changes the coupling (ratio of NADPH consumed versus product formed) for both arachidonic acid and palmitic acid oxidation. We have also analyzed cumene hydroperoxide-driven reactions and shown that they are unaffected by this mutation. These data, as well as fatty acid binding studies, support the hypothesis of a role of the I-helix residue, T268, in maintaining fatty acid substrates in the correct position for productive hydroxylation during the catalytic cycle of this enzyme.

References (42)

F.P. Guengerich
Trends Pharmacol. Sci.
(1991)
Y. Ishimura et al.
Biochem. Biophys. Res. Commun.
(1971)
T.L. Poulos et al.
J. Biol. Chem.
(1985)
Y. Kimata et al.
Biochem. Biophys. Res. Commun.
(1995)
T. Egawa et al.
Biochim. Biophys. Acta
(1990)
H. Furuya et al.
Biochem. Biophys. Res. Commun.
(1989)
K. Hiroya et al.
Arch. Biochem. Biophys.
(1994)
Y. Imai et al.
Biochem. Biophys. Res. Commun.
(1989)
Y. Imai et al.
FEBS Lett.
(1988)
Y. Miura et al.
Biochim. Biophys. Acta
(1975)

L.O. Narhi et al.

J. Biol. Chem.

(1986)

L.O. Narhi et al.

J. Biol. Chem.

(1987)

C.A. Hasemann et al.

Structure

(1995)

S.S. Boddupalli et al.

Arch. Biochem. Biophys.

(1992)

S.S. Boddupalli et al.

J. Biol. Chem.

(1990)

L.P. Wen et al.

J. Biol. Chem.

(1987)

T. Omura et al.

J. Biol. Chem.

(1964)

R.M. Laethem et al.

J. Biol. Chem.

(1992)

I. Sevrioukova et al.

Arch. Biochem. Biophys.

(1997)

J.H. Capdevila et al.

J. Biol. Chem.

(1996)

Cited by (49)

The full-length cytochrome P450 enzyme CYP102A1 dimerizes at its reductase domains and has flexible heme domains for efficient catalysis
2018, Journal of Biological Chemistry
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.
The role of cytochrome P450 BM3 phenylalanine-87 and threonine-268 in binding organic hydroperoxides
2016, Biochimica et Biophysica Acta - General Subjects
Citation Excerpt :
In another study [24], the T268A P450 BM3 mutant displayed either similar activity to WT or reduced activity depending on the length and the degree of branching of the fatty acid substrate. The T268A P450 BM3 mutant had lower activity toward specific substrates leading to the conclusion that the purpose of the threonine was to recognize and position the substrate in the active site [10,25]. Similar conclusions were made from the lower activity observed for mutations of T301 in rabbit CYP2C2 [19,26].
Cytochrome P450 (P450) BM3, from Bacillus megaterium, catalyzes a wide range of chemical reactions and is routinely used as a model system to study mammalian P450 reactions and structure.
The metabolism of 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHTOOH) and 2-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadien-1-one (BMPOOH) was examined with P450 BM3 and with the conserved T268 and F87 residues mutated to investigate their effects on organic hydroperoxide metabolism. To determine the effects of the mutations on the active site volume and architecture, the X-ray crystal structure of the F87A/T268A P450 BM3 heme domain (BMP) was determined and compared to previous structures. To investigate the interactions of the substrates with the F87 and T268 residues, BHTOOH and BMPOOH were docked into the BMP X-ray crystal structures.
Lower metabolism of BHTOOH and BMPOOH was observed in the WT P450 BM3 and the T268A P450 BM3 mutant than in the F87A and F87A/T268A P450 BM3 mutants. Large differences were found in the F–G loop regions and active site cavity volumes for the F87A mutated structures.
Analysis of the metabolism, X-ray crystal structures, and molecular docking simulations suggests that P450 BM3 activity toward BHTOOH and BMPOOH is mediated through substrate recognition by T268 and F87, and the active site cavity volume. Based on this information, a simplified representation is presented with the relative orientation of organic hydroperoxides in the P450 BM3 active site.
The metabolism results and structural analysis of this model P450 allowed us to rationalize the structural factors that influence organic hydroperoxide metabolism.
Cytochrome P450<inf>cin</inf> (CYP176A1) D241N: Investigating the role of the conserved acid in the active site of cytochrome P450s
2013, Biochimica et Biophysica Acta - Proteins and Proteomics
Citation Excerpt :
It has been reported that production of hydrogen peroxide in this case demonstrates the essential nature of the threonine in oxygen protonation in P450cam and is a specific example of uncoupling (NADH consumption without oxidised product formation) [13]. Subsequent studies with a range of eukaryotic and prokaryotic P450s have shown that mutation of the conserved threonine (or in some cases serine) often results in a significantly uncoupled enzyme [10,13–19]. Studies from several groups [7,15,20–26] also demonstrated that the conserved carboxylic acid (Asp251 in P450cam) was important in maintaining the rate but not specificity of oxygen activation and substrate oxidation.
P450_cin (CYP176A) is a rare bacterial P450 in that contains an asparagine (Asn242) instead of the conserved threonine that almost all other P450s possess that directs oxygen activation by the heme prosthetic group. However, P450_cin does have the neighbouring, conserved acid (Asp241) that is thought to be involved indirectly in the protonation of the dioxygen and affect the lifetime of the ferric-peroxo species produced during oxygen activation. In this study, the P450_cin D241N mutant has been produced and found to be analogous to the P450_cam D251N mutant. P450_cin catalyses the hydroxylation of cineole to give only (1R)-6β-hydroxycineole and is well coupled (NADPH consumed: product produced). The P450_cin D241N mutant also hydroxylated cineole to produce only (1R)-6β-hydroxycineole, was moderately well coupled (31 ± 3%) but a significant reduction in the rate of the reaction (2% as compared to wild type) was observed. Catalytic oxidation of a variety of substrates by D241N P450_cin were used to examine if typical reactions ascribed to the ferric-peroxo species increased as this intermediate is known to be more persistent in the P450_cam D251N mutant. However, little change was observed in the product profiles of each of these substrates between wild type and mutant enzymes and no products consistent with chemistry of the ferric-peroxo species were observed to increase.
Evaluation of structural features in fungal cytochromes P450 predicted to rule catalytic diversification
2013, Biochimica et Biophysica Acta - Proteins and Proteomics
Citation Excerpt :
Multiple sequence alignment of CYP61 enzymes with other P450s acting as C22-desaturases uncovered a unique common fragment, FLFA/SQDAS, accommodating a highly conserved alanine residue at position 340 in the Saccharomyces cerevisiae congener; this site was speculated to be generally vital to C22-desaturation processes [101]. Indeed, this I-helical A340 aligns with the hydrogen-bonding T268 in the bacterial CYP102A1 and equivalent threonines in most other P450s, known to have a key role in proper orientation of both substrate and the iron-bound distal oxygen atom to initiate O–O bond cleavage upon protonation [102]. Though alternative mechanisms of H+ delivery by a water molecule or a H-bonding machinery in a groove close to the oxygen-binding site have been discussed [103], the alanine for threonine exchange in CYP61A1 presently makes the precise way of H+ donation during O2 activation conjectural.
Fungi belong to the large kingdom of lower eukaryotic organisms encompassing yeasts along with filamentous and dimorphic members. Microbial P450 enzymes have contributed to exploration of and adaptation to diverse ecological niches such as conversion of lipophilic compounds to more hydrophilic derivatives or degradation of a vast array of environmental toxicants. To better understand diversification of the catalytic behavior of fungal P450s, detailed insight into the molecular machinery steering oxidative attack on the distinctly structured endogenous and xenobiotic substrates is of preeminent interest. Based on a general, CYP102A1-related template the bulk of predicted substrate/inhibitor-binding determinants were shown to cluster near the distal heme face within the six known substrate recognition sites (SRSs) made up by the α-helical B′/F/G/I tetrad, the B′–C interhelical loop and strands of the β6-sheet, population density being highest in the structurally flexible SRS-1 and SRS-4 domains, showing a low degree of conservation. Reactivity toward ligands favorably coincides with the lipophilicity/hydrophilicity profile and bulkiness of critical amino acids acting as selective filters. Some decisive elements may also serve in maintenance of catalytic competence via their action as gatekeepers directing substrate access/positioning or stabilizers of the heme environment enabling dioxygen activation. Non-SRS residues seem to control spin state equilibria and attract redox partners by electrostatic forces. Of note, the inhibitory potency of azole-type fungicides is likely to arise from perturbation of the complex interplay of the mechanistic principles addressed above. Knowledge-supported exploitation of the topological data will be helpful in the manufacture of commodity/specialty chemicals as well as therapeutic agents. Also, engineered fungal P450s may be used to improve pollutant-specific bioremediation of contaminated soils.
Interactions of cytochrome P450s with their ligands
2011, Archives of Biochemistry and Biophysics
Citation Excerpt :
As an interesting contrast, P450eryF (CYP107A1) exemplifies the importance of water-directing machinery for ligand binding and catalysis in a unique fashion. P450eryF lacks Thr268 essential for water positioning and oxygen binding for efficient turnover in P450 BM3, but harbors two similarly ordered water molecules in complex with its natural substrate 6-deoxyerythronolide B [50,51,53,54]. Nature has apparently accommodated a deficiency in P450eryF water-directing machinery through a high degree of substrate selectivity, in which the preferred substrate contains a hydroxyl group that directs water to the required locations.
Cytochrome P450s (CYPs) are heme-containing monooxygenases that contribute to an enormous range of enzymatic function including biosynthetic and detoxification roles. This review summarizes recent studies concerning interactions of CYPs with ligands including substrates, inhibitors, and diatomic heme-ligating molecules. These studies highlight the complexity in the relationship between the heme spin state and active site occupancy, the roles of water in directing protein–ligand and ligand–heme interactions, and the details of interactions between heme and gaseous diatomic CYP ligands. Both kinetic and thermodynamic aspects of ligand binding are considered.
Oxygen activation by P450<inf>cin</inf>: Protein and substrate mutagenesis
2011, Archives of Biochemistry and Biophysics
Citation Excerpt :
Mutagenesis of the conserved threonine in P450cam to alanine leads to a large increase in hydrogen peroxide production, with very little (approximately 5%) of the NADH-reducing equivalents used to produce the hydroxylated product [3,4]. Similar studies have been conducted on a range of other bacterial (e.g. P450BM-3) [5,6] and eukaryotic P450s (e.g. CYP1A2, 2B4, 2D6, 2E1) [7–10] and have yielded results consistent with those seen with P450cam. Based on these studies it has been concluded that this threonine is essential for catalytic activity.
A conserved threonine found in the majority of cytochromes P450 (P450s) has been implicated in the activation of dioxygen during the catalytic cycle. P450_cin (CYP176A) has been found to be an exception to this paradigm, where the conserved threonine has been replaced with an asparagine. Prior studies with a P450_cin N242A mutant established that the Asn-242 was not a functional replacement for the conserved threonine but was essential for the regio- and stereocontrol of the oxidation of cineole. To explore further how P450_cin controls the activation of the dioxygen in the absence of the conserved threonine, two concurrent lines of investigation were followed. Modification of P450_cin indicated that the Thr-243 was not involved in controlling the protonation of the hydroperoxy species. In addition, the N242T mutant did not enhance the rate and/or efficiency of catalytic turnover of cineole by P450_cin. In parallel experiments, the substrate cineole was modified by removing the ethereal oxygen to produce camphane or 2,2-dimethylbicyclo[2.2.2]octane (cinane). An analogous experiment with P450_EryF showed that a hydroxyl group on the substrate was vital, and in its absence catalytic turnover was effectively abolished. Catalytic turnover of P450_cin with either of these alternative substrates (camphane or cinane) revealed that in the absence of the ethereal oxygen there was still a significant amount of coupling of the NADPH-reducing equivalents to the formation of oxidised product. Again the substrate itself was not found to be important in controlling oxygen activation, in contrast to P450_EryF, but was shown to be essential for regio- and stereoselective substrate oxidation. Thus, it still remains unclear how dioxygen activation in the catalytic turnover of cineole by P450_cin is controlled.

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^☆: This work was supported in part by Grant R01-GM50858 from the NIH.

^☆☆: Ortiz de Montellano, P. R.

²: To whom correspondence should be addressed. Fax: (214) 648-8856. E-mail: [email protected].

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Regular ArticleThr268 in Substrate Binding and Catalysis in P450BM-3☆,☆☆

Abstract

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Regular Article
Thr268 in Substrate Binding and Catalysis in P450BM-3☆,☆☆