Protein engineering and mechanistic studies of morphinone reductase

The thesis describes mechanistic studies of the bacterial protein morphinone reductase (MR) using mutagenesis, kinetic and spectroscopic methods. The roles of seven active site residues were investigated. Cys-191 was initially predicted to be an active site acid; however, mutagenesis studies revealed that Cys-191 does not act in such role. His-186 and Asn-189 were shown to be involved in ligand binding. Mutation of His-186 to alanine has dramatic effects on the enzyme in the steady-state and in the oxidative half-reaction suggesting that His-186 could be an active site acid; however, pH dependence and NMR spectroscopic studies proved that His-186 could not play such role. No charge-transfer complex was observed between the H186A mutant and b-NADH in the reductive half-reaction. Mutation of Asn-189 affects enzyme kinetics and results in a slower rate of flavin oxidation. Faster rates for flavin reduction in the N189A mutant suggest that mutation of Asn-189 favours orientation of the flavin and b-NADH for hydride transfer. Neither Tyr-72 nor Tyr-356 is an active site acid. Mutation of Tyr-72 does not substantially affect enzyme kinetics. Mutations of Tyr-356 and Trp-106 had pronounced affects on enzyme kinetics, suggesting that each mutation alters the active site geometry. In the seven MR mutants studied, the flavin redox potentials are not altered compared to wild-type enzyme except for the T32A mutant where a 50 mV decrease is observed. Mutation of Thr-32 produced enhanced activity in the oxidative half-reaction and a slower activity in the reductive half-reaction effect of copper on MR was investigated. His-186 is shown to be involved in copper binding. 1-Nitrocyclohexene is a potential substrate for MR and the underlying mechanism requires further study

A biological route to conjugated alkenes: microbial production of hepta-1,3,5-triene

Conjugated alkenes such as dienes and polyenes have a range of applications as pharmaceutical agents and valuable building blocks in the polymer industry. Development of a renewable route to these compounds provides an alternative to fossil fuel derived production. The enzyme family of the UbiD decarboxylases offers substantial scope for alkene production, readily converting poly unsaturated acids. However, biochemical pathways producing the required substrates are poorly characterized, and UbiD-application has hitherto been limited to biological styrene production. Herein, we present a proof-of-principle study for microbial production of polyenes using a bioinspired strategy employing a polyketide synthase (PKS) in combination with a UbiD-enzyme. Deconstructing a bacterial iterative type II PKS enabled repurposing the broad-spectrum antibiotic andrimid biosynthesis pathway to access the metabolic intermediate 2,4,6-octatrienoic acid, a valuable chemical for material and pharmaceutical industry. Combination with the fungal ferulic acid decarboxylase (Fdc1) led to a biocatalytic cascade-type reaction for the production of hepta-1,3,5-triene in vivo. Our approach provides a novel route to generate unsaturated hydrocarbons and related chemicals and provides a blue-print for future development and application

Invisible hands Contract cleaning; a theological reflection

SIGLEAvailable from British Library Document Supply Centre-DSC:6224.18279(25) / BLDSC - British Library Document Supply CentreGBUnited Kingdo

A Biological Route to Conjugated Alkenes: Microbial Production of Hepta-1,3,5-triene

Conjugated alkenes such as dienes and polyenes have a range of applications as pharmaceutical agents and valuable building blocks in the polymer industry. Development of a renewable route to these compounds provides an alternative to fossil fuel derived production. The enzyme family of the UbiD decarboxylases offers substantial scope for alkene production, readily converting poly unsaturated acids. However, biochemical pathways producing the required substrates are poorly characterized, and UbiD-application has hitherto been limited to biological styrene production. Herein, we present a proof-of-principle study for microbial production of polyenes using a bioinspired strategy employing a polyketide synthase (PKS) in combination with a UbiD-enzyme. Deconstructing a bacterial iterative type II PKS enabled repurposing the broad-spectrum antibiotic andrimid biosynthesis pathway to access the metabolic intermediate 2,4,6-octatrienoic acid, a valuable chemical for material and pharmaceutical industry. Combination with the fungal ferulic acid decarboxylase (Fdc1) led to a biocatalytic cascade-type reaction for the production of hepta-1,3,5-triene in vivo. Our approach provides a novel route to generate unsaturated hydrocarbons and related chemicals and provides a blue-print for future development and application

KiPar, a tool for systematic information retrieval regarding parameters for kinetic modelling of yeast metabolic pathways

MOTIVATION: Most experimental evidence on kinetic parameters is buried in the literature, whose manual searching is complex, time consuming and partial. These shortcomings become particularly acute in systems biology, where these parameters need to be integrated into detailed, genome-scale, metabolic models. These problems are addressed by KiPar, a dedicated information retrieval system designed to facilitate access to the literature relevant for kinetic modelling of a given metabolic pathway in yeast. Searching for kinetic data in the context of an individual pathway offers modularity as a way of tackling the complexity of developing a full metabolic model. It is also suitable for large-scale mining, since multiple reactions and their kinetic parameters can be specified in a single search request, rather than one reaction at a time, which is unsuitable given the size of genome-scale models. RESULTS: We developed an integrative approach, combining public data and software resources for the rapid development of large-scale text mining tools targeting complex biological information. The user supplies input in the form of identifiers used in relevant data resources to refer to the concepts of interest, e.g. EC numbers, GO and SBO identifiers. By doing so, the user is freed from providing any other knowledge or terminology concerned with these concepts and their relations, since they are retrieved from these and cross-referenced resources automatically. The terminology acquired is used to index the literature by mapping concepts to their synonyms, and then to textual documents mentioning them. The indexing results and the previously acquired knowledge about relations between concepts are used to formulate complex search queries aiming at documents relevant to the user's information needs. The conceptual approach is demonstrated in the implementation of KiPar. Evaluation reveals that KiPar performs better than a Boolean search. The precision achieved for abstracts (60%) and full-text articles (48%) is considerably better than the baseline precision (44% and 24%, respectively). The baseline recall is improved by 36% for abstracts and by 100% for full text. It appears that full-text articles are a much richer source of information on kinetic data than are their abstracts. Finally, the combined results for abstracts and full text compared with the curated literature provide high values for relative recall (88%) and novelty ratio (92%), suggesting that the system is able to retrieve a high proportion of new documents