Identification of a new plasmid-encoded cytochrome P450 CYP107DY1 from Bacillus megaterium with a catalytic activity towards mevastatin

Milhim, Mohammed; Putkaradze, Natalia; Abdulmughni, Ammar; Kern, Fredy; Hartz, Philip; Bernhardt, Rita

doi:10.1016/j.jbiotec.2016.11.002

Cited by 14 publications

(12 citation statements)

References 46 publications

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“…However, the redox system BmCPR‐Fdx2 exhibited the highest efficiency, with 60% of peak recovery as compared with dithionite reduced CO‐difference peak. The redox partners BmCPR and Fdx2 originated from B. megaterium DAM319 [40] and have been shown to support efficiently the activity of CYP106A1 [21] and CYP107DY1 [22].…”

Section: Resultsmentioning

confidence: 99%

“…The capacity of the electron transfer partners was determined by comparing the peak at 450 nm of the CO-complex T2 reduced with the different redox systems and the peak at 450 nm of the CO-complex T2 reduced with sodium dithionite. P450-T2 was mixed with ferredoxins from Bacillus megaterium (Fdx2, Fdx3) [21,22], mammalian truncated adrenodoxin (Adx 4-108 ) [23,24] or yeast Etp1 [25] and corresponding ferredoxin reductases (BmCPR, AdR or Arh1) at a ratio of 1 : 40 : 5 (0.25 µM P450-T2: 10 µM Fdx: 1.25 µM FdR) in 50 mM HEPES buffer (pH 7.4). NADPH was added to achieve a final concentration of 1 mM.…”

Section: Redox Partner Screeningmentioning

confidence: 99%

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Characterization of a thermophilic cytochrome P450 of the CYP203A subfamily from Binh Chau hot spring in Vietnam

Nguyen

Milhim

et al. 2020

FEBS Open Bio

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Section: Resultsmentioning

confidence: 99%

Section: Redox Partner Screeningmentioning

confidence: 99%

Characterization of a thermophilic cytochrome P450 of the CYP203A subfamily from Binh Chau hot spring in Vietnam

Nguyen

Milhim

et al. 2020

FEBS Open Bio

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“…The diflavin reductase BmCPR and ferredoxin Fdx2 are electron transfer partners of Bacillus megaterium DSM319. The BmCPR-Fdx2 system efficiently supported the activity of CYP106A1 [31] and CYP107DY1 [32]. The bovine adrenodoxin reductase homologue 1 Arh1 and its natural redox partner Etp1 are originated from Schizosaccharomyces pombe [33].…”

Section: Identification Of Electron Transfer Partnersmentioning

confidence: 99%

A Novel Thermostable Cytochrome P450 from Sequence-Based Metagenomics of Binh Chau Hot Spring as a Promising Catalyst for Testosterone Conversion

Nguyen

Tung

et al. 2020

Catalysts

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Biotechnological applications of cytochromes P450 show difficulties, such as low activity, thermal and/or solvent instability, narrow substrate specificity and redox partner dependence. In an attempt to overcome these limitations, an exploitation of novel thermophilic P450 enzymes from nature via uncultured approaches is desirable due to their great advantages that can resolve nearly all mentioned impediments. From the metagenomics library of the Binh Chau hot spring, an open reading frame (ORF) encoding a thermostable cytochrome P450—designated as P450-T3—which shared 66.6% amino acid sequence identity with CYP109C2 of Sorangium cellulosum So ce56 was selected for further identification and characterization. The ORF was synthesized artificially and heterologously expressed in Escherichia coli C43(DE3) using the pET17b system. The purified enzyme had a molecular weight of approximately 43 kDa. The melting temperature of the purified enzyme was 76.2 °C and its apparent half-life at 60 °C was 38.7 min. Redox partner screening revealed that P450-T3 was reduced well by the mammalian AdR-Adx4-108 and the yeast Arh1-Etp1 redox partners. Lauric acid, palmitic acid, embelin, retinoic acid (all-trans) and retinoic acid (13-cis) demonstrated binding to P450-T3. Interestingly, P450-T3 also bound and converted testosterone. Overall, P450-T3 might become a good candidate for biocatalytic applications on a larger scale.

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“…Class BmCPR is known to transfer electrons to Etp1 fd and various Fds but also supports the reaction of the microsomal CYP21A2 very efficiently without any Fd mediator [134,135]. In contrast, CaCPR1 supports activity with microsomal CYP1A2 only at low rates relative to the cognate mammalian cytochrome P450 reductase [136].…”

Section: Thermostable Diflavin Reductasesmentioning

confidence: 99%

Determinants of thermostability in the cytochrome P450 fold

Harris

Thomson

Strohmaier

et al. 2018

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Cytochromes P450 are found throughout the biosphere in a wide range of environments, serving a multitude of physiological functions. The ubiquity of the P450 fold suggests that it has been co-opted by evolution many times, and likely presents a useful compromise between structural stability and conformational flexibility. The diversity of substrates metabolized and reactions catalyzed by P450s makes them attractive starting materials for use as biocatalysts of commercially useful reactions. However, process conditions impose different requirements on enzymes to those in which they have evolved naturally. Most natural environments are relatively mild, and therefore most P450s have not been selected in Nature for the ability to withstand temperatures above ~40°C, yet industrial processes frequently require extended incubations at much higher temperatures. Thus, there has been considerable interest and effort invested in finding or engineering thermostable P450 systems. Numerous P450s have now been identified in thermophilic organisms and analysis of their structures provides information as to mechanisms by which the P450 fold can be stabilized. In addition, protein engineering, particularly by directed or artificial evolution, has revealed mutations that serve to stabilize particular mesophilic enzymes of interest. Here we review the current understanding of thermostability as it applies to the P450 fold, gleaned from the analysis of P450s characterized from thermophilic organisms and the parallel engineering of mesophilic forms for greater thermostability. We then present a perspective on how this information might be used to design stable P450 enzymes for industrial application. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

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Identification of a new plasmid-encoded cytochrome P450 CYP107DY1 from Bacillus megaterium with a catalytic activity towards mevastatin

Cited by 14 publications

References 46 publications

Characterization of a thermophilic cytochrome P450 of the CYP203A subfamily from Binh Chau hot spring in Vietnam

Characterization of a thermophilic cytochrome P450 of the CYP203A subfamily from Binh Chau hot spring in Vietnam

A Novel Thermostable Cytochrome P450 from Sequence-Based Metagenomics of Binh Chau Hot Spring as a Promising Catalyst for Testosterone Conversion

Determinants of thermostability in the cytochrome P450 fold

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