Engineering substrate recognition in catalysis by cytochrome P450cam

Bell, Stephen; Chen, X.; Feng, Xu; Rao, Zihe; Wong, Luet‐Lok

doi:10.1042/bst0310558

Cited by 33 publications

(24 citation statements)

References 42 publications

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“…Regardless of the area of application, however, the feasibility of utilizing P450s to confer herbicide resistance or decontaminate polluted areas will greatly depend on our abilities to provide P450 enzymes with high catalytic activity towards the desired substrate(s). Several approaches have been successful in enhancing P450 activity including rational protein design (Bell et al 2003;Carmichael and Wong 2001), directed enzyme evolution (Abecassis et al 2000;Abecassis et al 2003) and the optimization of the redox environment in plants by simultaneously overexpressing a NADPH:P450 reductase (Siminszky et al 2003); therefore, P450s specifically tailored for high efficiency herbicide metabolism may soon become available.…”

Section: Practical Applicationsmentioning

confidence: 99%

Plant cytochrome P450-mediated herbicide metabolism

2006

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In the last two decades it has become apparent that enzymes of the P450 monooxygenase (P450) superfamily are responsible for the Phase I metabolism of numerous herbicides representing several classes of organic compounds. The majority of experimental evidence for P450 involvement in herbicide metabolism has been derived from in vitro studies in which the catalytic activity of plant microsomes towards herbicidal substrates was measured in the presence of various P450 inhibitors and activators. While the studies with microsomes elicited much appreciation for the pivotal roles of plant P450s in herbicide metabolism, detailed characterization of these enzymes only became possible after the isolation of genes encoding specific isoforms responsible for herbicide conversion. Several lines of evidence suggest that the development of herbicide resistance in weeds by enhanced detoxification is frequently associated with elevated levels of P450 activity. Enhanced detoxification-based herbicide resistance is particularly difficult to control, because it can involve resistance to multiple, chemically unrelated classes of herbicides. Continued research efforts are aimed at elucidating the role of P450s in the metabolic fates of herbicides in plants and the development of herbicide resistance in weeds. Recent advances made in the isolation and genetic manipulation of P450 enzymes have created new opportunities for their application in engineering herbicide tolerance and bioremediation.

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Section: Practical Applicationsmentioning

confidence: 99%

Plant cytochrome P450-mediated herbicide metabolism

2006

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“…1A). Alternatively, NysL can be engineered via either site-specific mutagenesis (4) or directed evolution (3) in order to change its specificity towards 10-deoxynystatin. This approach might provide novel nystatin analogues hydroxylated at alternative macrolactone ring positions in hope of obtaining antifungal compounds with improved pharmacologic properties.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the P450 Monooxygenase NysL, Responsible for C-10 Hydroxylation during Biosynthesis of the Polyene Macrolide Antibiotic Nystatin in Streptomyces noursei

Volokhan

Sletta

Ellingsen

et al. 2006

Appl Environ Microbiol

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The nysL gene, encoding a putative P450 monooxygenase, was identified in the nystatin biosynthetic gene cluster of Streptomyces noursei. Although it has been proposed that NysL is responsible for hydroxylation of the nystatin precursor, experimental evidence for this activity was lacking. The nysL gene was inactivated in S. noursei by gene replacement, and the resulting mutant was shown to produce 10-deoxynystatin. Purification and an in vitro activity assay for 10-deoxynystatin demonstrated its antifungal activity being equal to that of nystatin. The NysL protein was expressed heterologously in Escherichia coli as a His-tagged protein and used in an enzyme assay with 10-deoxynystatin as a substrate. The results obtained clearly demonstrated that NysL is a hydroxylase responsible for the post-polyketide synthase modification of 10-deoxynystatin at position C-10. Kinetic studies with the purified recombinant enzyme allowed determination of K m and k cat and revealed no inhibition of recombinant NysL by either the substrate or the product. These studies open the possibility for in vitro evolution of NysL aimed at changing its specificity, thereby providing new opportunities for engineered biosynthesis of novel nystatin analogues hydroxylated at alternative positions of the macrolactone ring.

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“…Most studies of these enzymes focus on altering the specificity and regioselectivity of the catalyzed hydroxylation reactions. Analysis of the active site of CYP101A1 and comparisons of the structure of target substrates with that of camphor led to the design of active-site mutants with greatly enhanced activity for the oxidation of (+)-α-pinene [30]. Based on the enzyme/ substrate contacts revealed by the structure of the triple mutant F87W/Y96F/V247L with (+)-α-pinene bound within the active site, additional mutations were carried out and resulted in an improved selectivity for the formation of the natural fragrances and flavors (+)-cis-verbenol and (+)-verbenone [31].…”

Section: Engineering By Rational Protein Designmentioning

confidence: 99%

Terpene Hydroxylation with Microbial Cytochrome P450 Monooxygenases

Janocha

Schmitz

Bernhardt

2015

Biotechnology of Isoprenoids

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Terpenoids comprise a highly diverse group of natural products. In addition to their basic carbon skeleton, they differ from one another in their functional groups. Functional groups attached to the carbon skeleton are the basis of the terpenoids' diverse properties. Further modifications of terpene olefins include the introduction of acyl-, aryl-, or sugar moieties and usually start with oxidations catalyzed by cytochrome P450 monooxygenases (P450s, CYPs). P450s are ubiquitously distributed throughout nature, involved in essential biological pathways such as terpenoid biosynthesis as well as the tailoring of terpenoids and other natural products. Their ability to introduce oxygen into nonactivated C-H bonds is unique and makes P450s very attractive for applications in biotechnology. Especially in the field of terpene oxidation, biotransformation methods emerge as an attractive alternative to classical chemical synthesis. For this reason, microbial P450s depict a highly interesting target for protein engineering approaches in order to increase selectivity and activity, respectively. Microbial P450s have been described to convert industrial and pharmaceutically interesting terpenoids such as ionones, limone, valencene, resin acids, and triterpenes (including steroids) as well as vitamin D3. Highly selective and active mutants have been evolved by applying classical site-directed mutagenesis as well as directed evolution of proteins. As P450s usually depend on electron transfer proteins, mutagenesis has also been applied to improve the interactions between P450s and their respective redox partners. This chapter provides an overview of terpenoid hydroxylation reactions catalyzed by bacterial P450s and highlights the achievements made by protein engineering to establish productive hydroxylation processes.

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Engineering substrate recognition in catalysis by cytochrome P450cam

Cited by 33 publications

References 42 publications

Plant cytochrome P450-mediated herbicide metabolism

Plant cytochrome P450-mediated herbicide metabolism

Characterization of the P450 Monooxygenase NysL, Responsible for C-10 Hydroxylation during Biosynthesis of the Polyene Macrolide Antibiotic Nystatin in Streptomyces noursei

Terpene Hydroxylation with Microbial Cytochrome P450 Monooxygenases

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