The self-sufficient CYP102 family enzyme, Krac9955, from Ktedonobacter racemifer DSM44963 acts as an alkyl- and alkyloxy-benzoic acid hydroxylase

Maddigan, Natasha K.; Bell, Stephen

doi:10.1016/j.abb.2016.12.014

Cited by 14 publications

(4 citation statements)

References 35 publications

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“…Unraveling the details underlying the unique catalytic features of CYP505D6 and Krac9955 will require further clarification by site-directed mutagenesis and three-dimensional structural studies. Compared with CYP102A1 and CYP505A1, CYP505D6 and Krac9955 show low activity toward saturated fatty acids with the same carbon chain length (56). The poor regioselectivity of the fatty acid hydroxylation resulting from a large entrance might cause low catalytic activity.…”

Section: Discussionmentioning

confidence: 99%

Biochemical Characterization of CYP505D6, a Self-Sufficient Cytochrome P450 from the White-Rot Fungus Phanerochaete chrysosporium

Sakai

Matsuzaki

Wise

et al. 2018

Appl Environ Microbiol

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The activity of a self-sufficient cytochrome P450 enzyme CYP505D6 from the lignin-degrading basidiomycete was characterized. Recombinant CYP505D6 was produced in and purified. In the presence of NADPH, CYP505D6 used a series of saturated fatty alcohols with C9-18 carbon chain lengths as substrates. Hydroxylation occurred at ω-1 to ω-6 positions of such substrates with C9-15 carbon chain lengths, except for 1-dodecanol, which was hydroxylated at ω-1 to ω-7 positions. Fatty acids were also substrates of CYP505D6. Based on the sequence alignment, the corresponding amino acid of Tyr51, which is located at the entrance to the active site pocket in CYP102A1, was Val51 in CYP505D6. To understand the diverse hydroxylation mechanism, wild-type CYP505D6 and its V51Y variant, and wild-type CYP102A1 and its Y51V variant were generated, and the products of their reaction with dodecanoic acid analyzed. Compared with the wild-type CYP505D6, its V51Y variant generated fewer products hydroxylated at ω-4 to ω-6 positions. The products generated by the wild-type CYP102A1 were hydroxylated at ω-1 to ω-4 positions, whereas its Y51V variant generated ω-1 to ω-7 hydroxydodecanoic acids. These observations indicated that Val51 plays an important role in determining the regio-specificity of fatty acid hydroxylation, at least that at ω-4 to ω-6 positions. Aromatic compounds, such as naphthalene and 1-naphthol, were also hydroxylated by CYP505D6. These findings highlight a unique broad substrate spectrum of CYP505D6, rendering it an attractive candidate enzyme for the biotechnological industry. is a white-rot fungus whose metabolism of lignin, aromatic pollutants, and lipids has been most extensively studied. This fungus harbors 154 cytochrome P450-encoding genes in the genome. As evidenced in the current study, CYP505D6, a fused protein of P450 and its reductase, hydroxylates fatty alcohols (C9-15) and fatty acids (C9-15) at ω-1 to ω-7 or ω-6 positions, respectively. Naphthalene and 1-naphthol were also hydroxylated, indicating that the substrate specificity of CYP505D6 is broader than that of the known fused proteins CYP102A1 and CYP505A1. Substrate versatility of CYP505D6 makes this enzyme an attractive candidate for biotechnological applications.

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Section: Discussionmentioning

confidence: 99%

Biochemical Characterization of CYP505D6, a Self-Sufficient Cytochrome P450 from the White-Rot Fungus Phanerochaete chrysosporium

Sakai

Matsuzaki

Wise

et al. 2018

Appl Environ Microbiol

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“…Aliquots of substrate solution (1–5 μL) were added (from a 25 or 100 mM stock solution) until no further change in the spectrum was observed. The HS (high-spin) content of the substrate-bound enzyme was determined using the method previously described by Maddigan and Bell …”

Section: Methodsmentioning

confidence: 99%

“…The HS (high-spin) content of the substratebound enzyme was determined using the method previously described by Maddigan and Bell. 69 Heme Bleaching Assay. Heme bleaching experiments were performed according to the previously published procedure.…”

Section: Production and Purification Of Hazakqementioning

confidence: 99%

Engineering Peroxygenase Activity into Cytochrome P450 Monooxygenases through Modification of the Oxygen Binding Region

Podgorski,

Akter,

Churchman

et al. 2024

ACS Catal.

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Cytochrome P450 enzymes (CYPs) are biocatalysts for the generation of fine chemicals including natural products, drug metabolites, and flavor and fragrance compounds. However, both the high cost of the required nicotinamide cofactors and their need for additional electron transfer proteins limit their use. Here, we investigate whether CYPs can be converted into more efficient peroxygenases through protein engineering of the enzyme's oxygen activation machinery. We improve the peroxygenase activity by modifying selected residues within the I-helix to more closely resemble those of a natural peroxygenase. We produced mutants containing two, four, and six mutations, within this region of the Ihelix. In our model CYP system, the double mutant in which glutamine and glutamate residues replaced aspartate and threonine, respectively, was found to have significantly higher peroxygenase activity for the O-demethylation of 4-methoxybenzoic acid than a single glutamate mutant prototype. Importantly, it functioned better at lower H 2 O 2 concentrations and could convert all the added substrate to product. All the mutants maintained the stereoselectivity of the CYP enzyme for the epoxidation of 4-vinylbenzoic acid. The X-ray crystal structures of these enzymes showed significant structural changes at the oxygen-binding groove in the I-helix. In crystallo reactions with 4-methylbenzoic acid exhibit electron density corresponding to the 4-(hydroxymethyl)benzoic acid metabolite. We extended this mutagenesis strategy to a bacterial steroid-hydroxylating CYP and an uncharacterized CYP from a thermophilic bacterium. In these instances, we generate peroxygenases, which catalyze the regio-and stereoselective hydroxylation of progesterone and the hydroxylation of fatty acids at low hydrogen peroxide concentrations.

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“…Substrate binding: spin state determination and binding titrations : The high‐spin heme content was determined using an enzyme concentration of ∼2–3 μM in 50 mM Tris, pH 7.4, with the addition of 0.5–1 μL aliquots of substrate (from a 100 mM stock) until the spectrum did not change. The percentage shift was estimated (to approximately ±5%) as described previously [11c,24] …”

Section: Methodsmentioning

confidence: 99%

Exploring the Factors which Result in Cytochrome P450 Catalyzed Desaturation Versus Hydroxylation

Coleman

Doherty

Zhang

et al. 2022

Chemistry An Asian Journal

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The cytochrome P450 family of monooxygenase enzymes have essential biological roles involving the selective oxidation of carbon‐hydrogen bonds. They can also catalyze other important metabolic reactions including desaturation to form alkenes. Currently the factors that control the partition between P450 hydroxylation and desaturation pathways are poorly defined. The CYP199A4 enzyme from the bacterium Rhodopseudomonas palustris HaA2 catalyzes the oxidation of 4‐ethyl‐ and 4‐isopropyl‐ benzoic acids with hydroxylation and desaturation occurring in significant quantities. Here we demonstrate that 4‐cyclopropylbenzoic acid is regioselectively hydroxylated by CYP199A4 at the benzylic carbon. In contrast, the oxidation of 4‐n‐propylbenzoic acid by CYP199A4 results in three major metabolites: an alkene from desaturation and two hydroxylation products at the benzylic (Cα) and Cβ carbons in similar quantities. Extending the length of the alkyl substituent resulted in 4‐n‐butylbenzoic acid being oxidized at the benzylic position (45%) and desaturated (55%). In contrast, 4‐isobutylbenzoic generated very little alkene (5%) but was hydroxylated at the benzylic position (54%) and at the tertiary Cβ position (41%). The oxidation of 4‐n‐propylbenzoic acid by the F298 V mutant of CYP199A4 occurred with no hydroxylation at Cβ and a significant increase in metabolites arising from desaturation (73%). The X‐ray crystal structures of CYP199A4 with each substrate revealed that they bind in the active site with the alkyl substituent positioned over the heme. However, the longer alkylbenzoic acids were bound in a different conformation as was 4‐n‐propylbenzoic acid in the F298 V mutant. Overall, the changes in metabolite distribution could be ascribed to bond strength differences and the position of the alkyl group relative to the heme.

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The self-sufficient CYP102 family enzyme, Krac9955, from Ktedonobacter racemifer DSM44963 acts as an alkyl- and alkyloxy-benzoic acid hydroxylase

Cited by 14 publications

References 35 publications

Biochemical Characterization of CYP505D6, a Self-Sufficient Cytochrome P450 from the White-Rot Fungus Phanerochaete chrysosporium

Biochemical Characterization of CYP505D6, a Self-Sufficient Cytochrome P450 from the White-Rot Fungus Phanerochaete chrysosporium

Engineering Peroxygenase Activity into Cytochrome P450 Monooxygenases through Modification of the Oxygen Binding Region

Exploring the Factors which Result in Cytochrome P450 Catalyzed Desaturation Versus Hydroxylation

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