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Purification and Characterization of Rhodobacter sphaeroides Polyhistidine-tagged HemA and Comparison with Purified Polyhistidine-tagged HemT

Xiao, Xiao, Mr.
http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1371650467

Abstract Details

2013, Master of Science (MS), Bowling Green State University, Biological Sciences.
All tetrapyrrole, molecules that include heme, bacteriochlorophyll, and vitamin B12, are derived from 5-aminolevulinic acid (ALA). In the purple non-sulfur alphaproteobacteria Rhodobacter sphaeroides ALA is formed by the condensation of glycine and succinyl-CoA, catalyzed by the pyridoxal-phosphate dependent enzyme ALA synthase. Two ALA synthase genes, hemA and hemT are present in R. sphaeroides wild type strain 2.4.1. When expressed, either one of the gene products can satisfy the ALA requirement of the cell. Towards understanding the presence of two ALA synthases in one organism, each enzyme should be characterized individually in order to define what is similar and different about the enzymes. Using this information, one may be able to infer how the activities of the two ALA synthases are coordinate in R. sphaeroides. In this study, R. sphaeroides 2.4.1 recombinant polyhistidine-tagged HemA (rHemA) was affinity purified and its optimum temperature and pH, specific activity, and kinetic properties were determined. The effect of added hemin on its activity was also evaluated, as was its secondary structure composition using circular dichroism. These characteristics were then compared to those of recombinant polyhistidine-tagged HemT (rHemT). Two major differences were noted. First, the catalytic capacity of rHemA is more than ten times greater than rHemT. Second, rHemA has a higher affinity for succinyl-CoA than rHemT. A hypothesis that could explain the significance of these differences posits that HemT is needed to reduce ALA synthesis under conditions in which succinyl-CoA and glycine are in greater demand for metabolisms other than ALA formation, such as energy generation for the former and protein synthesis for the latter. Examining hemA and hemT expression under conditions that would impose such metabolic priorities would be appropriate to test this hypothesis.
Jill Zeilstra-Ryalls, Dr. (Advisor)
Scott Rogers, Dr. (Committee Member)
Zhaohui Xu, Dr. (Committee Member)
44 p.
Microbiology
ALA synthase; purification

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Citations

  • Xiao, X. (2013). Purification and Characterization of Rhodobacter sphaeroides Polyhistidine-tagged HemA and Comparison with Purified Polyhistidine-tagged HemT [Master's thesis, Bowling Green State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1371650467

    APA Style (7th edition)

  • Xiao, Xiao. Purification and Characterization of Rhodobacter sphaeroides Polyhistidine-tagged HemA and Comparison with Purified Polyhistidine-tagged HemT . 2013. Bowling Green State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1371650467.

    MLA Style (8th edition)

  • Xiao, Xiao. "Purification and Characterization of Rhodobacter sphaeroides Polyhistidine-tagged HemA and Comparison with Purified Polyhistidine-tagged HemT ." Master's thesis, Bowling Green State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1371650467

    Chicago Manual of Style (17th edition)

bgsu1371650467
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This open access ETD is published by Bowling Green State University and OhioLINK.