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Man Tsz-Kwong

Advisor: Malcolm Winkler, Ph.D.

Vitamin B6 (or pyridoxal 5′-phosphate, PLP) is an essential, ubiquitous coenzyme that affects many aspects of amino acid and cellular metabolism in all organisms. The goal of this thesis is to examine the regulation of PLP biosynthesis in Escherichia coli K-12. First, PdxH oxidase is a PLP biosynthetic enzyme, which uses molecular oxygen as an electron acceptor under aerobic assay conditions. To test if facultative anaerobic E. coli uses another enzyme to replace the function of PdxH oxidase anaerobically, suppressors of a pdxH null mutant were isolated anaerobically after 2-aminopurine or spontaneous mutagenesis. Only one specific bypass mutation in another PLP biosynthetic gene pdxJ was found, suggesting that PdxH oxidase is able to function anaerobically and PdxT utilizes D-1-deoxyxyulose as a substrate. Second, regulation of the serC (pdxF)-aroA operon, which is involved the biosynthesis of L-serine, PLP and aromatic compounds was examined. A serC (pdxF) single gene transcript and a serC (pdXf)-aroA cotranscript initiated at P{serC\ (pdxF)} upstream of serC (pdxF) were detected. The expression of the operon is activated by leucine responsive regulatory protein (LRP) and repressed by cAMP receptor protein-cAMP complex (CRP-cAMP) at the transcriptional level. LRP activates the operon by directly binding to the upstream consensus box. Binding of CRP-cAMP to the upstream CRP box diminishes the activation effect of LRP. However, deletion of the CRP box did not affect the repression of CRP-cAMP, suggesting that CRP-cAMP may repress the operon indirectly by stimulating the activity or level of an unidentified repressor. The overall effect of this regulation is to maximize the expression of the operon when the cells are growing in minimal-glucose medium. In addition, the binding and the transcription of P{serC\ (pdxF)} by RNA polymerase require a supercoiled circular DNA, indicating that DNA supercoiling affects the transcription of the operon. Third, regulation of another PLP biosynthetic gene gapB was also examined. gapB is activated by CRP-cAMP and repressed by catabolic repressor activator protein (CRA). However, the activation of CRP-cAMP is epistatic to the repression of CRA. Due to the CRA repression, gapB was expressed at a low level in all the media tested, suggesting that it may be the rate-limiting step of PLP biosynthesis. In summary, unlike genes in many biosynthetic pathways, PLP biosynthetic genes are regulated by global regulators that are important for carbon and amino acid metabolism, instead of the end product(s) of the pathway.

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Research Info

Regulation of Pyridoxal 5′-phosphate biosynthesis in Escherichia coli K-12