RESUMO
The cytochrome P450 monooxygenases (P450s) catalyze a vast array of oxygenation reactions that can be useful in biocatalytic applications. CYP101J2 from Sphingobium yanoikuyae is a P450 that catalyzes the hydroxylation of 1,8-cineole. Here we report the crystallization and X-ray structure elucidation of recombinant CYP101J2 to 1.8 Å resolution. The CYP101J2 structure shows the canonical P450-fold and has an open conformation in the absence of substrate. Analysis of the structure revealed that CYP101J2, in the absence of substrate, forms a well-ordered substrate-binding channel that suggests a unique form of substrate guidance in comparison to other bacterial 1,8-cineole-hydroxylating P450 enzymes. Proteins 2017; 85:945-950. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas de Bactérias/química , Cicloexanóis/química , Sistema Enzimático do Citocromo P-450/química , Monoterpenos/química , Sphingomonadaceae/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Cicloexanóis/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Eucaliptol , Expressão Gênica , Hidroxilação , Modelos Moleculares , Monoterpenos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sphingomonadaceae/enzimologia , Especificidade por SubstratoRESUMO
We report the isolation and characterization of three new cytochrome P450 monooxygenases: CYP101J2, CYP101J3, and CYP101J4. These P450s were derived from Sphingobium yanoikuyae B2, a strain that was isolated from activated sludge based on its ability to fully mineralize 1,8-cineole. Genome sequencing of this strain in combination with purification of native 1,8-cineole-binding proteins enabled identification of 1,8-cineole-binding P450s. The P450 enzymes were cloned, heterologously expressed (N-terminally His6 tagged) in Escherichia coli BL21(DE3), purified, and spectroscopically characterized. Recombinant whole-cell biotransformation in E. coli demonstrated that all three P450s hydroxylate 1,8-cineole using electron transport partners from E. coli to yield a product putatively identified as (1S)-2α-hydroxy-1,8-cineole or (1R)-6α-hydroxy-1,8-cineole. The new P450s belong to the CYP101 family and share 47% and 44% identity with other 1,8-cineole-hydroxylating members found in Novosphingobium aromaticivorans and Pseudomonas putida Compared to P450cin (CYP176A1), a 1,8-cineole-hydroxylating P450 from Citrobacter braakii, these enzymes share less than 30% amino acid sequence identity and hydroxylate 1,8-cineole in a different orientation. Expansion of the enzyme toolbox for modification of 1,8-cineole creates a starting point for use of hydroxylated derivatives in a range of industrial applications. IMPORTANCE: CYP101J2, CYP101J3, and CYP101J4 are cytochrome P450 monooxygenases from S. yanoikuyae B2 that hydroxylate the monoterpenoid 1,8-cineole. These enzymes not only play an important role in microbial degradation of this plant-based chemical but also provide an interesting route to synthesize oxygenated 1,8-cineole derivatives for applications as natural flavor and fragrance precursors or incorporation into polymers. The P450 cytochromes also provide an interesting basis from which to compare other enzymes with a similar function and expand the CYP101 family. This could eventually provide enough bacterial parental enzymes with similar amino acid sequences to enable in vitro evolution via DNA shuffling.
Assuntos
Cânfora 5-Mono-Oxigenase/isolamento & purificação , Cânfora 5-Mono-Oxigenase/metabolismo , Cicloexanóis/metabolismo , Monoterpenos/metabolismo , Esgotos/microbiologia , Sphingomonadaceae/enzimologia , Biotransformação , Cânfora 5-Mono-Oxigenase/classificação , Cânfora 5-Mono-Oxigenase/genética , Citrobacter/enzimologia , Citrobacter/genética , Transporte de Elétrons , Escherichia coli/genética , Eucaliptol , Genoma Bacteriano , Hidroxilação , Microbiologia Industrial , Ligação Proteica , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Proteínas Recombinantes/metabolismo , Sphingomonadaceae/genética , Sphingomonadaceae/isolamento & purificação , Sphingomonadaceae/metabolismoRESUMO
In the simultaneous saccharification and fermentation to ethanol of 100 g l(-1) microcrystalline cellulose, the cellobiose-fermenting recombinant Klebsiella oxytoca P2 outperformed a range of cellobiose-fermenting yeasts used in earlier work, despite producing less ethanol than reported earlier for this organism under similar conditions. The time taken by K. oxytoca P2 to produce up to about 33 g l(-1) ethanol was much less than for any other organism investigated, including ethanol-tolerant strains of Saccharomyces pastorianus, Kluyveromyces marxianus and Zymomonas mobilis. Ultimately, it produced slightly less ethanol (maximum 36 g l(-1)) than these organisms, reflecting its lower ethanol tolerance. Significant advantages were obtained by co-culturing K. oxytoca P2 with S. pastorianus, K. marxianus or Z. mobilis, either isothermally, or in conjunction with temperature-profiling to raise the cellulase activity. Co-cultures produced significantly more ethanol, more rapidly, than either of the constituent strains in pure culture at the same inoculum density. K. oxytoca P2 dominated the early stages of the co-cultures, with ethanol production in the later stages due principally to the more ethanol tolerant strain. The usefulness of K. oxytoca P2 in cellulose simultaneous saccharification and fermentation should be improved by mutation of the strain to increase its ethanol tolerance.
