Recombinant expression and purification of the 2,5-diketocamphane 1,2-monooxygenase from the camphor metabolizing Pseudomonas putida strain NCIMB 10007

Three different Baeyer-Villiger monooxygenases (BVMOs) were reported to be involved in the camphor metabolism by Pseudomonas putida NCIMB 10007. During (+)-camphor degradation, 2,5-diketocamphane is formed serving as substrate for the 2,5-diketocamphane 1,2-monooxygenase. This enzyme is encoded on the CAM plasmid and depends on the cofactors FMN and NADH and hence belongs to the group of type II BVMOs. We have cloned and recombinantly expressed the oxygenating subunit of the 2,5-diketocamphane 1,2-monooxygenase (2,5-DKCMO) in E. coli followed by His-tag-based affinity purification. A range of compounds representing different BVMO substrate classes were then investigated, but only bicyclic ketones were converted by 2,5-DKCMO used as crude cell extract or after purification. Interestingly, also (-)-camphor was oxidized, but conversion was about 3-fold lower compared to (+)-camphor. Moreover, activity of purified 2,5-DKCMO was observed in the absence of an NADH-dehydrogenase subunit

Baeyer-Villiger monooxygenases involved in camphor degradation

In this thesis, all three BVMOs from Pseudomonas putida NCIMB10007, that were known to be responsible for the ability of this strain to degrade camphor since the 1950s were successfully made available as recombinant biocatalysts. While the genomic sequence of 2,5-DKCMO was available from the database, the genes encoding 3,6-DKCMO and OTEMO had to be identified using certain PCR-techniques first. All three enzymes were cloned into standard plasmids enabling convenient expression in E. coli facilitating the application of the enzymes in organic chemistry. Their synthetic potential was already reported during the 1990s, but at that time their efficient application was limited due to difficulties with respect to low production levels and insufficient purity and separation of enzyme fractions. These drawbacks are now overcome. Furthermore, biochemical characterization of the camphor-degrading BVMOs was performed including the substrate spectra of these enzymes. Thereby OTEMO turned out not only to have a broad substrate scope accepting mono- and bicyclic aliphatic and arylaliphatic ketones, but also to efficiently convert alpha/beta-unsaturated cycloalkanones due to the similarity of these compounds to OTEMOs natural substrate. Finally, the major limitation in the synthetic application of Type II BVMOs was addressed by searching a flavin-reductase suitable for coupling to these two-component oxygenases. Putative candidates from the respective P. putida strain were identified by the use of amino acid motifs conserved in other representatives of two-component systems. While these enzymes failed, flavin-reductase Fre from E. coli - that also contained the motifs - was shown to enhance the activity of the DKCMOs when applied as crude cell extract as well as pure enzyme. This finding represents a key step for future application of Type II BVMOs.In dieser Doktorarbeit ist es gelungen, alle drei BVMOs aus Pseudomonas putida NCIMB 10007, von denen seit den 1950er Jahren bekannt ist, dass sie am mikrobiellen Abbau von Camper beteiligt sind, als rekombinante Biokatalysatoren zur Verfügung zu stellen. Während die genomische Sequenz der 2,5-DKCMO in der NCBI-Datenbank erhältlich war, mussten die Gene, die die 3,6-DKCMO und die OTEMO kodieren zunächst mit Hilfe von PCR-Techniken identifiziert werden. Alle drei Enzyme wurden anschließend in Standard-Plasmide kloniert, die ihre Expression in E. coli erlaubten. Dies sollte die Anwendung der Enzyme in der organischen Chemie ermöglichen. Das synthetische potential dieser Enzyme wurde bereits in den 1990er Jahren untersucht, aber damals war ihre effiziente Anwendung durch Schwierigkeiten bezüglich ihrer Herstellung in größeren Mengen und zufriedenstellender Reinheit, sowie ausreichender Trennung der Enzyme voneinander, limitiert. Diese Schwierigkeiten sind nun überwunden. Weiterhin wurde die biochemische Charakterisierung der Campher-abbauenden BVMOs durchgeführt, einschließlich der Substrat-Spektren der Enzyme. Dabei zeigt such, dass OTEMO nicht nur ein breites Spektrum an Substraten wie mono- und bizyklische, aliphatische und arylaliphatische Ketone akzeptiert. Weiterhin setzte sie effizient alpha/beta-ungesättigte Zykloalkanone um, die Ähnlichkeiten zum natürlichen Substrat des Enzyms aufweisen. Schließlich widmete sich diese Doktorarbeit der größten Einschränkung in der Anwendung von Typ II BVMOs, der Notwendigkeit einer zusätzlichen Flavin-Reduktase zur Versorgung der Oxygenase mit reduziertem Flavin. Zunächst wurden mit Hilfe von Aminosäuremotiven, die in anderen Zwei-Komponenten-Monooxygenase-Systhemen konserviert sind, Flavin-Reduktasen aus P. putida identifiziert. Diese Enzyme waren jedoch nicht geeignet, um mit den DKCMOs zusammenzuarbeiten. Stattdessen wurde die Flavin-Reduktase Fre aus E. coli, die ebenfalls die genannten Motive enthält, verwendet, um die Aktivität der DKCMOs gegenüber ihren Substraten zu steigern. Dies wurde in dieser Arbeit sowohl für die Verwendung von Enzym-Rohextrakten als auch aufgereinigten Enzymen gezeigt und stellt einen wichtigen Schritt in der zukünftigen Anwendung der Typ II BVMOs dar

Functional assembly of camphor converting two-component Baeyer-Villiger monooxygenases with a flavin reductase from E-coli

The major limitation in the synthetic application of two-component Baeyer-Villiger monooxygenases was addressed by identifying the 28-kDa flavin-reductase Fre from Escherichia coli as a suitable supplier of reduced FMN for these enzymes. Coexpression of Fre with either 2,5- or 3,6-diketocamphane monooxygenase from Pseudomonas putida NCIMB 10007 significantly enhanced the conversion of camphor and norcamphor serving as representative ketones. With purified enzymes, full conversion was achieved, while only slight amounts of product were formed in the absence of this flavin reductase. Fusion of the genes of Fre and DKCMOs into single open reading frame constructs resulted in unstable proteins exhibiting flavin reducing, but poor oxygenating activity, which led to overall decreased conversion of camphor.AuthorCount:5;</p

A general protein purification and immobilization method on controlled porosity glass : biocatalytic applications

A general combined purification and immobilization method to facilitate biocatalytic process development is presented. The support material, EziG (TM), is based on controlled porosity glass (CPG) or polymer-coated versions thereof (HybCPG) and binds protein affinity tags. Biocatalytic reactions in aqueous and organic media with seven enzymes of biocatalytic interest are shown.AuthorCount:7;</p

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: The first crystal structure of a type II Baeyer-Villiger monooxygenase

The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9\uc5 resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a \u3b2-bulge at the C-terminus of \u3b2-strand 3, which is a feature observed in many proteins of this superfamily.Peer reviewed: YesNRC publication: Ye

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: The first crystal structure of a type II Baeyer-Villiger monooxygenase

The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9\uc5 resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a \u3b2-bulge at the C-terminus of \u3b2-strand 3, which is a feature observed in many proteins of this superfamily.Peer reviewed: YesNRC publication: Ye

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer-Villiger monooxygenase. Corrigendum

: A statement is amended in the article by Isupov et al. [(2015). Acta Cryst. D71, 2344-2353]