Structure‐Guided Redesign of CYP153A<sub><i>M.aq</i></sub> for the Improved Terminal Hydroxylation of Fatty Acids

Hoffmann, Sara M.; Danesh-Azari, Hamid-Reza; Spandolf, Claudia; Weissenborn, Martin J.; Grogan, Gideon; Hauer, Bernhard

doi:10.1002/cctc.201600680

Cited by 21 publications

(35 citation statements)

References 45 publications

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“…Dodecanoic acid is predicted to interact with a similar set of residues: Leu92 (SRS1); Leu176 (SRS2); Phe238, Leu239, Val242, Ala243, Thr247 (SRS4); Ala290, Pro289 (SRS5) and Ile395 (SRS6). In such a predicted binding conformation the C7, C8, C9, C10 and C11 carbon atoms are most optimally located for hydroxylation ( Figure 6B [65], which are highly regioselective, display a narrow hydrophobic tunnel for binding FAs, restricting substrate motility in their active site. Likely, all in-chain FA hydroxylating P450s display similar active site properties as CYP267B1, explaining why so far no 'exclusive P450 in-chain hydroxylase' has been reported [52].…”

Section: Cyp267b1-tetradecanoic Acid Complex and Prediction Of Fatty mentioning

confidence: 99%

Structural insights into oxidation of medium-chain fatty acids and flavanone by myxobacterial cytochrome P450 CYP267B1

Jozwik

Litzenburger

Khatri

et al. 2018

Biochemical Journal

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Oxidative biocatalytic reactions performed by cytochrome P450 enzymes (P450s) are of high interest for the chemical and pharmaceutical industries. CYP267B1 is a P450 enzyme from myxobacterium So ce56 displaying a broad substrate scope. In this work, a search for new substrates was performed, combined with product characterization and a structural analysis of substrate-bound complexes using X-ray crystallography and computational docking. The results demonstrate the ability of CYP267B1 to perform in-chain hydroxylations of medium-chain saturated fatty acids (decanoic acid, dodecanoic acid and tetradecanoic acid) and a regioselective hydroxylation of flavanone. The fatty acids are mono-hydroxylated at different in-chain positions, with decanoic acid displaying the highest regioselectivity towardsω-3 hydroxylation. Flavanone is preferably oxidized to 3-hydroxyflavanone. High-resolution crystal structures of CYP267B1 revealed a very spacious active site pocket, similarly to other P450s capable of converting macrocyclic compounds. The pocket becomes more constricted near to the heme and is closed off from solvent by residues of the F and G helices and the B-C loop. The crystal structure of the tetradecanoic acid-bound complex displays the fatty acid bound near to the heme, but in a nonproductive conformation. Molecular docking allowed modeling of the productive binding modes for the four investigated fatty acids and flavanone, as well as of two substrates identified in a previous study (diclofenac and ibuprofen), explaining the observed product profiles. The obtained structures of CYP267B1 thus serve as a valuable prediction tool for substrate hydroxylations by this highly versatile enzyme and will encourage future selectivity changes by rational protein engineering.

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Section: Cyp267b1-tetradecanoic Acid Complex and Prediction Of Fatty mentioning

confidence: 99%

Structural insights into oxidation of medium-chain fatty acids and flavanone by myxobacterial cytochrome P450 CYP267B1

Jozwik

Litzenburger

Khatri

et al. 2018

Biochemical Journal

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“…In 2016, the structure of CYP153A from Marinobacter aquaeolei (CYP153A M.aq ) was solved with and without its 12-hydroxydodecanoic acid ligand. The authors found that CYP153A M.aq has terminal hydroxylase activity and that the A231G single mutation induced three-fold higher activity by increasing the flexibility of the substrate binding region [16]. Previous studies also demonstrated that Sphingomonas sp.…”

Section: Introductionmentioning

confidence: 99%

Crystal Structure of a Putative Cytochrome P450 Alkane Hydroxylase (CYP153D17) from Sphingomonas sp. PAMC 26605 and Its Conformational Substrate Binding

Lee

et al. 2016

IJMS

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Enzymatic alkane hydroxylation reactions are useful for producing pharmaceutical and agricultural chemical intermediates from hydrocarbons. Several cytochrome P450 enzymes catalyze the regio- and stereo-specific hydroxylation of alkanes. We evaluated the substrate binding of a putative CYP alkane hydroxylase (CYP153D17) from the bacterium Sphingomonas sp. PAMC 26605. Substrate affinities to C10–C12 n-alkanes and C10–C14 fatty acids with Kd values varied from 0.42 to 0.59 μM. A longer alkane (C12) bound more strongly than a shorter alkane (C10), while shorter fatty acids (C10, capric acid; C12, lauric acid) bound more strongly than a longer fatty acid (C14, myristic acid). These data displayed a broad substrate specificity of CYP153D17, hence it was named as a putative CYP alkane hydroxylase. Moreover, the crystal structure of CYP153D17 was determined at 3.1 Å resolution. This is the first study to provide structural information for the CYP153D family. Structural analysis showed that a co-purified alkane-like compound bound near the active-site heme group. The alkane-like substrate is in the hydrophobic pocket containing Thr74, Met90, Ala175, Ile240, Leu241, Val244, Leu292, Met295, and Phe393. Comparison with other CYP structures suggested that conformational changes in the β1–β2, α3–α4, and α6–α7 connecting loop are important for incorporating the long hydrophobic alkane-like substrate. These results improve the understanding of the catalytic mechanism of CYP153D17 and provide valuable information for future protein engineering studies.

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“…Such a strategy of modulating substrate specificity has been successfully demonstrated on various fatty acid utilizing enzymes in literature. [12] We also based the selection of some of our mutants on recent studies with FAP, where mutations were generally performed for other purposes than our aim here. The list of mutants and the purpose in their selection are given in Table 1.…”

Section: Selection Of Mutation Sitesmentioning

confidence: 99%

Optimization and Engineering of Fatty Acid Photodecarboxylase for Substrate Specificity

et al. 2021

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Fatty acid photodecarboxylase (FAP) is one of the few photoenzymes in nature. The ability of FAP to convert fatty acids into alka(e)nes without the need for reducing equivalents put this enzyme into spotlight for biocatalytic applications. Although it has been discovered only a few years ago, many studies already emerged demonstrating its potential in areas from biofuel production and enzymatic kinetic resolution to being a critical component of multi-enzyme cascades. While there have been few protein engineering studies for modulating activity of FAP towards very short chain fatty acids, no study has yet addressed substrate selectivity within the medium to long chain fatty acid range, where FAP shows great promise for the synthesis of drop-in biofuels from ubiquitous fatty acids with chain lengths from C12 to C18. Here, after determining optimum expression and assay conditions for FAP, we screened 22 rationally designed mutant enzymes towards four naturally abundant fatty acid substrates; C12 : 0, C16 : 0, C18 : 0 and C18 : 1. Depending on the type of the exchanged amino acid, we observed selectivity shifts towards shorter or longer chains, compared to wild type enzyme. Notably, we obtained two groups of mutants; one group with high selectivity towards only C18 : 0, and another group that is selective towards C12 : 0 substrate. Moreover, we measured light and thermal stability of the wild type enzyme as well as the light stability of a mutant engineered for selectivity.

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Structure‐Guided Redesign of CYP153A_M.aq for the Improved Terminal Hydroxylation of Fatty Acids

Cited by 21 publications

References 45 publications

Structural insights into oxidation of medium-chain fatty acids and flavanone by myxobacterial cytochrome P450 CYP267B1

Structural insights into oxidation of medium-chain fatty acids and flavanone by myxobacterial cytochrome P450 CYP267B1

Crystal Structure of a Putative Cytochrome P450 Alkane Hydroxylase (CYP153D17) from Sphingomonas sp. PAMC 26605 and Its Conformational Substrate Binding

Optimization and Engineering of Fatty Acid Photodecarboxylase for Substrate Specificity

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Structure‐Guided Redesign of CYP153AM.aq for the Improved Terminal Hydroxylation of Fatty Acids

Cited by 21 publications

References 45 publications

Structural insights into oxidation of medium-chain fatty acids and flavanone by myxobacterial cytochrome P450 CYP267B1

Structural insights into oxidation of medium-chain fatty acids and flavanone by myxobacterial cytochrome P450 CYP267B1

Crystal Structure of a Putative Cytochrome P450 Alkane Hydroxylase (CYP153D17) from Sphingomonas sp. PAMC 26605 and Its Conformational Substrate Binding

Optimization and Engineering of Fatty Acid Photodecarboxylase for Substrate Specificity

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Structure‐Guided Redesign of CYP153A_M.aq for the Improved Terminal Hydroxylation of Fatty Acids