Coenzyme Q (ubiquinone) biosynthesis in Escherichia coli : isolation and characterization of ubiF mutant

Includes bibliographical references (pages [32]-36).Coenzyme Q (ubiquinone) plays an essential role in aerobic electron transport in Escherichia coli. It is involved in electron transfer and proton translocation in the electron transport chains of mitochondria, bacteria and cyanobacteria. In E. coli, the Q biosynthetic pathway consists of nine enzymatic reactions in which steps four, six, and eight are hydroxylation reactions and steps five, seven, and nine are methylation reactions. The genes ubiB, ubiH, and ubiF encode the hydroxylases. The ubiF gene maps at 14.87 minutes of the E. coli chromosome. The enzyme it encodes is responsible for the conversion of 2-octaprenyl-3-methyl- 6-methoxy-l,4-benzoquinol to 2-octaprenyl-3-methyl-5- hydroxy-6-methoxy-l,4-benzoquinol and mutants unable to undergo this conversion accumulate the precursor. orf391, which has amino acid similarity to another hydroxylase encoding gene (ubiH), was cloned into a multi-copy plasmid. Complementation of the ubiF mutant, JF496, with orf391 restored the strain's ability to grow using a reduced substrate, such as succinate as the main carbon source. Also, accumulation of an intermediate eluted from a thin layer chromatogram showed a spectra identical to the one previously reported for the intermediate 2-octaprenyl-3-methyl-6-methoxy-l,4- benzoquinol. From this data, orf391 is identified as the ubiF gene.M.S. (Master of Science