The Impact of Linker Length on P450 Fusion Constructs: Activity, Stability and Coupling

Hoffmann, Sara M.; Weissenborn, Martin J.; Gricman, Łukasz; Notonier, Sandra; Pleiss, Jürgen; Hauer, Bernhard

doi:10.1002/cctc.201501397

Cited by 29 publications

(31 citation statements)

References 40 publications

(98 reference statements)

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“…It can catalyze enantio‐ and regioselective hydroxylations, and it consists of two catalytic domains: the CPR, which is a NADPH‐dependent FAD‐ and FMN‐containing reductase domain, and a monooxygenase heme domain. In some studies, various artificial reductase–monooxygenase fusions were made, either to improve stability and/or to improve electron‐coupling efficiency . Sabbadin et al.…”

Section: Enzyme Fusions In Biocatalysismentioning

confidence: 99%

Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes

2018

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One approach to bringing enzymes together for multienzyme biocatalysis is genetic fusion. This enables the production of multifunctional enzymes that can be used for whole-cell biotransformations or for in vitro (cascade) reactions. In some cases and in some aspects, such as expression and conversions, the fused enzymes outperform a combination of the individual enzymes. In contrast, some enzyme fusions are greatly compromised in activity and/or expression. In this Minireview, we give an overview of studies on fusions between two or more enzymes that were used for biocatalytic applications, with a focus on oxidative enzymes. Typically, the enzymes are paired to facilitate cofactor recycling or cosubstrate supply. In addition, different linker designs are briefly discussed. Although enzyme fusion is a promising tool for some biocatalytic applications, future studies could benefit from integrating the findings of previous studies in order to improve reliability and effectiveness.

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Section: Enzyme Fusions In Biocatalysismentioning

confidence: 99%

Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes

2018

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“…This is a great improvement on growing cells where the product concentration and the productivity were 0.08 g L −1 and 2.6 mg L −1 h −1 respectively under equal bioreactor conditions and similar optical densities. P450 RhF constructs in the corresponding cell lysates were analyzed by CO difference spectroscopy using excess NADPH as the reducing agent (Page S3, Supporting information) . This spectrum was compared to a traditional P450 CO difference spectrum and showed equivalent absorbance at 450 nm, indicating correct fusion and function of the election delivering partners with the haem domain of P450 RhF (Table S6, Supporting information).…”

Section: Resultsmentioning

confidence: 99%

The self‐sufficient P450 RhF expressed in a whole cell system selectively catalyses the 5‐hydroxylation of diclofenac

Klenk

Nebel

Porter

et al. 2017

Biotechnology Journal

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Abbreviations: ALA, aminolevulinic acid; BM3, cytochrome P450 from Bacillus megaterium; cdw, cell dry weight; cww, cell wet weight; HPLC, high--performance liquid chromatography; IMAC, immobilised metal affinity chromatography; IPTG, isopropyl β--D--1--thiogalactopyranoside; IR, infrared; MS, mass spectrometry; NADPH, nicotinamide adenine dinucleotide phosphate; NMR, nuclear magnetic resonance; NSAID, non--steroidal anti--inflammatory drug; P450, cytochrome P450 monooxygenase; RhF, cytochrome P450 from Rhodococcus sp. strain NCIMB 9784. 4Abstract P450 monooxygenases are able to catalyse the highly regio--and stereoselective oxidations of many organic molecules. However, the scale--up of such bio--oxidations remains challenging due to the often--low activity, level of expression and stability of P450 biocatalysts. Despite these challenges they are increasingly desirable as recombinant biocatalysts, particularly for the production of drug metabolites. Diclofenac is a widely used anti--inflammatory drug that is persistent in the environment along with the 4'-- and 5--hydroxy metabolites. Here we have used the self--sufficient P450 RhF (CYP116B2) from Rhodococcus sp. in a whole cell system to reproducibly catalyse the highly regioselective oxidation of diclofenac to 5--hydroxydiclofenac. The product is a human metabolite and as such is an important standard for environmental and toxicological analysis. Furthermore, access to significant quantities of 5--hydroxydiclofenac has allowed us to demonstrate further oxidative degradation to the toxic quinoneimine product. Our studies demonstrate the potential for gram--scale production of human drug metabolites through recombinant whole cell biocatalysis. 5

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“…This comparison suggested that residues 1-23 probably functioned as the membrane anchor and should be dispensable in terms of catalytic activity, so N-terminal trimming was carried out as shown in Figure 6. The linker determines the interaction between the P450 and BMR domains thus facilitating electron flow [69][70][71] and reducing its length by just six residues can abolish the activity of the fusion enzyme [49]. We therefore chose a linker containing 29 amino acids.…”

Section: Cloning Strategymentioning

confidence: 99%

Strategies for the construction of insect P450 fusion enzymes

Talmann

Wiesner

Vilcinskas

2017

Zeitschrift Für Naturforschung C

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Cytochrome P450 monooxygenases (P450s) are ubiquitous enzymes with a broad substrate spectrum. Insect P450s are known to catalyze reactions such as the detoxification of insecticides and the synthesis of hydrocarbons, which makes them useful for many industrial processes. Unfortunately, it is difficult to utilize P450s effectively because they must be paired with cytochrome P450 reductases (CPRs) to facilitate electron transfer from reduced nicotinamide adenine dinucleotide phosphate (NADPH). Furthermore, eukaryotic P450s and CPRs are membrane-anchored proteins, which means they are insoluble and therefore difficult to purify when expressed in their native state. Both challenges can be addressed by creating fusion proteins that combine the P450 and CPR functions while eliminating membrane anchors, allowing the production and purification of soluble multifunctional polypeptides suitable for industrial applications. Here we discuss several strategies for the construction of fusion enzymes combining insect P450 with CPRs.

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The Impact of Linker Length on P450 Fusion Constructs: Activity, Stability and Coupling

Cited by 29 publications

References 40 publications

Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes

Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes

The self‐sufficient P450 RhF expressed in a whole cell system selectively catalyses the 5‐hydroxylation of diclofenac

Strategies for the construction of insect P450 fusion enzymes

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