Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation

Joo, Hyun; Lin, Zhanglin; Arnold, Frances H.

doi:10.1038/21395

Cited by 422 publications

(236 citation statements)

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“…For random mutagenesis, the vao gene in pBC11 was amplified using manganese-based error-prone PCR mutagenesis (34,35). 25 l of a PCR mixture containing 10 pmol of primers 5Ј-GTA-AAACGACGGCCAGT-3Ј and 5Ј-CAGGAAACAGCTATGAC-3Ј, 90 ng of template DNA, and 250 or 500 pmol of MnCl 2 was heated for 3 min at 95°C and, after cooling down to 80°C, mixed with 25 l of a mixture containing 0.4 mM each dATP and dGTP, 2 mM each dCTP and dTTP, 2.5 units of Taq DNA polymerase, 20 mM Tris-HCl (pH 8.85), 4 mM MgCl 2 , 50 mM KCl, and 10 mM (NH 4 ) 2 SO 4 in a thin-wall PCR tube.…”

Section: Methodsmentioning

confidence: 99%

Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

Heuvel

Berg

Rovida

et al. 2004

Journal of Biological Chemistry

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The flavoenzyme vanillyl-alcohol oxidase was subjected to random mutagenesis to generate mutants with enhanced reactivity to creosol (2-methoxy-4-methylphenol). The vanillyl-alcohol oxidase-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) is oxidized to the widely used flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde). The first step of this reaction is extremely slow due to the formation of a covalent FAD N-5-creosol adduct. After a single round of error-prone PCR, seven mutants were generated with increased reactivity to creosol. The single-point mutants I238T, F454Y, E502G, and T505S showed an up to 40-fold increase in catalytic efficiency (k cat /K m ) with creosol compared with the wild-type enzyme. This enhanced reactivity was due to a lower stability of the covalent flavin-substrate adduct, thereby promoting vanillin formation. The catalytic efficiencies of the mutants were also enhanced for other ortho-substituted 4-methylphenols, but not for p-cresol (4-methylphenol). The replaced amino acid residues are not located within a distance of direct interaction with the substrate, and the determined three-dimensional structures of the mutant enzymes are highly similar to that of the wild-type enzyme. These results clearly show the importance of remote residues, not readily predicted by rational design, for the substrate specificity of enzymes.The increased use of enzymes and other proteins in the pharmaceutical, chemical, and agricultural industry has generated considerable interest in the design of proteins with new or improved properties. Two different but complementary technologies have been applied to this goal: (i) rational design, which relies on the availability of the three-dimensional structure and knowledge about the relationship between sequence, structure, and mechanism, and (ii) directed evolution methods, which use random mutagenesis of the gene encoding the protein or recombination of gene fragments to create diversity and then experimental screening of the libraries generated for the desired properties. Rational design has been used to elucidate and change enzyme mechanism, substrate and product specificity, enantioselectivity, cofactor specificity, and protein stability (1-3). Directed evolution has been applied to increase catalytic activity; to invert or improve enantioselectivity; and to alter substrate and product specificity, protein stability, pH optimum, and tolerance against organic solvents (1, 4 -7). An obvious advantage of directed evolution methods over sitedirected mutagenesis is that enzymes can be tailored for the production of (intermediate) products without detailed knowledge of protein structure and structure-function relationships.In this study, we engineered the flavoenzyme vanillyl-alcohol oxidase (VAO) 1 by random mutagenesis such that the evolved mutants are capable of producing natural vanillin (4-hydroxy-3-methoxybenzaldehyde) from the precursor creosol (2-methoxy...

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Section: Methodsmentioning

confidence: 99%

Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

Heuvel

Berg

Rovida

et al. 2004

Journal of Biological Chemistry

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“…This method is, however, associated with the problem of fast inactivation of the enzyme. However, directed evolution has been successfully applied to evolve the heme domains of P450cam and P450 BM-3 to enhance the efficiency of the "peroxide shunt" pathway (Joo et al 1999;Cirino and Arnold 2002).…”

Section: Direct Chemical Reduction Of the Heme Iron Is Very Inefficiementioning

confidence: 99%

“…Using error-prone PCR some mutants of P450cam (Joo et al 1999) and P450 BM-3 (Cirino and Arnold 2003) were created, which demonstrate high activity with hydrogen peroxide, which acts as both an oxidant and an electron donor, thereby substituting NAD(P)H.…”

Section: Protein Engineering Of P450 Monooxygenasesmentioning

confidence: 99%

Microbial P450 enzymes in biotechnology

Urlacher

Lutz-Wahl

Schmid

2004

Applied Microbiology and Biotechnology

200

100

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Oxidations are key reactions in chemical syntheses. Biooxidations using fermentation processes have already conquered some niches in industrial oxidation processes, since they allow the introduction of oxygen even into non-activated carbon atoms in a sterically and optically selective manner which is difficult or impossible to achieve by synthetic organic chemistry. Biooxidation using isolated enzymes is limited to oxidases and dehydrogenases. In addition, P450-catalyzed reactions require a constant supply of NAD(P)H which makes continuous cell-free processes very expensive. Quite recently, several groups have started to investigate cost-efficient ways which could allow the continuous supply of electrons to the heme iron. These include, for example, the use of electron mediators, direct electron supply from electrodes and enzymatic approaches. In addition, methods of protein design and directed evolution have been applied in an attempt to enhance the activity of the enzymes and improve their selectivity. The promising application of bacterial P450s as catalyzing agents in biocatalytic reactions and recent progress made in this field are covered in this review.3

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“…Other alternatives have explored the use of alternative electron sources; however turnovers of such systems are typically much lower than with NAD(P)H [51]. A more radical approach has been suggested, which exploits the capability of some CYPs to work as peroxygenases (EC 1.11.2.4) while performing the desired reaction [53]. Indeed, there are some P450 enzymes that catalyze the monooxygenation reaction without any electron-transferring co-factors in the presence of either hydrogen peroxide or an organic peroxide such as cumene hydroperoxide [54].…”

Section: Exploring Complex Enzymatic Mechanisms: P450 Monooxygenase Amentioning

confidence: 99%

A probabilistic framework for the exploration of enzymatic capabilities based on feasible kinetics and control analysis

Saa

Nielsen

2016

Biochimica et Biophysica Acta (BBA) - General Subjects

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BackgroundAnalysis of limiting steps within enzyme-catalyzed reactions is fundamental to understand their behavior and regulation. Methods capable of unravelling control properties and exploring kinetic capabilities of enzymatic reactions would be particularly useful for protein and metabolic engineering. While single-enzyme control analysis formalism has previously been applied to well-studied enzymatic mechanisms, broader application of formalism is limited in practice by the limited amount of kinetic data and the difficulty of describing complex allosteric mechanisms. MethodsTo overcome these limitations, we present here a probabilistic framework enabling control analysis of previously unexplored mechanisms under uncertainty. By combining a thermodynamically consistent parameterization with an efficient Sequential Monte Carlosampler embedded in a Bayesian setting, this framework yields insights into the capabilities of enzyme-catalyzed reactions with modest kinetic information, provided that the catalytic mechanism and a thermodynamic reference point are defined. ResultsThe framework was used to unravel the impact of thermodynamic affinity, substrate saturation levels and effector concentrations on the flux control and response coefficients of a diverse set of enzymatic reactions. ConclusionsOur results highlight the importance of the metabolic context in the control analysis of isolated enzymes as well as the use of statistically sound methods for their interpretation. General SignificanceThis framework significantly expands our current capabilities for unravelling the control properties of general reaction kinetics with limited amount of information. This framework will be useful for both theoreticians and experimentalists in the field.

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Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation

Cited by 422 publications

References 28 publications

Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

Laboratory-evolved Vanillyl-alcohol Oxidase Produces Natural Vanillin

Microbial P450 enzymes in biotechnology

A probabilistic framework for the exploration of enzymatic capabilities based on feasible kinetics and control analysis

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