A tale of two reductases: extending the bacteriochlorophyll biosynthetic pathway in E. coli

Autor: Maureen B. Quin, Ilya B. Tikh, Claudia Schmidt-Dannert
Jazyk: angličtina
Rok vydání: 2014
Předmět:
Applied Microbiology
lcsh:Medicine
Reductase
medicine.disease_cause
Biochemistry
Protein Structure
Secondary
Substrate Specificity
Energy-Producing Processes
Chlorobi
chemistry.chemical_compound
Bioreactors
Engineering
Protein structure
Microbial Physiology
Photosynthesis
lcsh:Science
Bacteriochlorophylls
Heme
chemistry.chemical_classification
Multidisciplinary
biology
Microbial Growth and Development
Recombinant Proteins
Cytochemistry
Synthetic Biology
Genetic Engineering
Oxidoreductases
Oxidation-Reduction
Protein Binding
Research Article
Biotechnology
Molecular Sequence Data
Bioengineering
Rhodobacter sphaeroides
Bioenergetics
Microbiology
Bacterial Proteins
Biosynthesis
Escherichia coli
medicine
Amino Acid Sequence
Biology
Microbial Metabolism
Organisms
Genetically Modified
lcsh:R
biology.organism_classification
Biosynthetic Pathways
Enzyme
chemistry
lcsh:Q
Bacteriochlorophyll
Zdroj: PLoS ONE, Vol 9, Iss 2, p e89734 (2014)
PLoS ONE
ISSN: 1932-6203
Popis: The creation of a synthetic microbe that can harvest energy from sunlight to drive its metabolic processes is an attractive approach to the economically viable biosynthetic production of target compounds. Our aim is to design and engineer a genetically tractable non-photosynthetic microbe to produce light-harvesting molecules. Previously we created a modular, multienzyme system for the heterologous production of intermediates of the bacteriochlorophyll (BChl) pathway in E. coli. In this report we extend this pathway to include a substrate promiscuous 8-vinyl reductase that can accept multiple intermediates of BChl biosynthesis. We present an informative comparative analysis of homologues of 8-vinyl reductase from the model photosynthetic organisms Rhodobacter sphaeroides and Chlorobaculum tepidum. The first purification of the enzymes leads to their detailed biochemical and biophysical characterization. The data obtained reveal that the two 8-vinyl reductases are substrate promiscuous, capable of reducing the C8-vinyl group of Mg protoporphyrin IX, Mg protoporphyrin IX methylester, and divinyl protochlorophyllide. However, activity is dependent upon the presence of chelated Mg(2+) in the porphyrin ring, with no activity against non-Mg(2+) chelated intermediates observed. Additionally, CD analyses reveal that the two 8-vinyl reductases appear to bind the same substrate in a different fashion. Furthermore, we discover that the different rates of reaction of the two 8-vinyl reductases both in vitro, and in vivo as part of our engineered system, results in the suitability of only one of the homologues for our BChl pathway in E. coli. Our results offer the first insights into the different functionalities of homologous 8-vinyl reductases. This study also takes us one step closer to the creation of a nonphotosynthetic microbe that is capable of harvesting energy from sunlight for the biosynthesis of molecules of choice.
Databáze: OpenAIRE
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