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Purification and Characterization of Rhodobacter sphaeroides 2.4.1 HemT and Comparison with HemA Isoenzyme

Kaganjo, James Chege
http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1371650426

Abstract Details

2013, Master of Science (MS), Bowling Green State University, Biological Sciences.
Rhodobacter sphaeroides synthesizes heme, vitamin B12 and bacteriochlorophyll to support its metabolic versatility that includes the ability to obtain energy by chemo- and phototrophy. The common precursor of all tetrapyrroles is 5-aminolevulinic acid (ALA) which in these bacteria is formed from the condensation of glycine and succinyl-CoA, a reaction catalyzed by PLP-dependent ALA synthase. ALA synthase is a member of the class II of fold type I PLP-dependent enzymes that also includes 2-amino-3-ketobutyrate-CoA ligase (KBL), serine palmitoyltransferase (SPT) and 8-amino-7-oxononanoate synthase (AONS). In R. sphaeroides strain 2.4.1, there are two ALA synthase genes, hemA and hemT, and when expressed each one alone can satisfy the requirement for ALA under all conditions that have been examined. In order to understand the role of the two ALA synthase isoenzymes, purification, characterization and comparison of the characteristics of both isoenzymes is necessary. In this study, recombinant polyhistidine-tagged HemT (rHemT) was purified using nickel affinity chromatography, and it was characterized in terms of optimum pH and temperature, the effect of added hemin, its kinetic properties, its secondary structure, the presence of disulfide bonds, and its activity using an alternative substrate. The properties of rHemT were then compared to those of recombinant polyhistidine-tagged HemA (rHemA), which was analyzed separately. The specific activity of rHemT was more than 10-fold lower than that of rHemA. The glycine Km for both proteins was the same, although the catalytic turnover rate for glycine was more than 10-fold higher for rHemA than for rHemT. The succinyl-CoA Km for rHemA was 2-fold lower than for rHemT, and again the catalytic turnover was 10-fold higher for rHemA than for rHemT. These differences indicate that HemT is a low activity ALA synthase when compared to HemA, which would be explained if HemT has a greater preference for substrates other than succinyl-CoA. Towards this end, the ability of rHemT to use acetyl-CoA was evaluated. Using purified rHemT no detectable product was formed, indicating that succinyl-CoA is the preferred substrate. Together with the comparisons of the HemA and HemT characteristics, a consideration of the substrates used in ALA production has suggested a possible explanation for the need to have both enzymes. This relies on the hypotheses that, (1) when hemA and hemT are both expressed, the products are able to form heterodimeric proteins, and (2) such heterodimers are less active than HemA homodimers, although possibly more active than HemT homodimers; i.e. hemT behaves in a dominant negative fashion to hemA. If true, then the role of HemT could be to reduce ALA synthase activity in the cell when succinyl-CoA and/or glycine are required for energy metabolism and protein synthesis, respectively.
Jill Zeilstra-Ryalls (Advisor)
Scott Rogers (Committee Member)
Raymond Larsen (Committee Member)
56 p.
Microbiology

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Citations

  • Kaganjo, J. C. (2013). Purification and Characterization of Rhodobacter sphaeroides 2.4.1 HemT and Comparison with HemA Isoenzyme [Master's thesis, Bowling Green State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1371650426

    APA Style (7th edition)

  • Kaganjo, James. Purification and Characterization of Rhodobacter sphaeroides 2.4.1 HemT and Comparison with HemA Isoenzyme. 2013. Bowling Green State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1371650426.

    MLA Style (8th edition)

  • Kaganjo, James. "Purification and Characterization of Rhodobacter sphaeroides 2.4.1 HemT and Comparison with HemA Isoenzyme." Master's thesis, Bowling Green State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1371650426

    Chicago Manual of Style (17th edition)

bgsu1371650426
912
© 2013, some rights reserved.Creative Commons License Purification and Characterization of Rhodobacter sphaeroides 2.4.1 HemT and Comparison with HemA Isoenzyme by James Chege Kaganjo is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by Bowling Green State University and OhioLINK.