5-Aminolevulinic acid biosynthesis in Escherichia coli.
Item
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Title
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5-Aminolevulinic acid biosynthesis in Escherichia coli.
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Identifier
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AAI9807914
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identifier
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9807914
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Creator
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Chen, Wei.
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Contributor
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Advisers: C. S. Russell | S. D. Cosloy
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Date
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1997
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry | Biology, Molecular | Biology, Genetics
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Abstract
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In E. coli, heme biosynthesis is regulated at the level of synthesis of its first committed precursor, 5-aminolevulinic acid (ALA). ALA is synthesized by the C{dollar}\sb5{dollar} pathway. Glutamate is converted to glutamyl-tRNA by glutamyl-tRNA synthetase (GTS). Glutamyl-tRNA is reduced to glutamyl-semialdehyde (GSA) by glutamyl-tRNA reductase (GTR), the gene product of hemA. GSA is converted to ALA by GSA aminotransferase. This last step also takes place non-enzymatically. Because glutamyl-tRNA is used for protein synthesis as well, regulation of ALA synthesis is probably at the level of GTR synthesis or activity.;hemM, which is located 213 bp up stream from hemA and transcribed divergently, was assumed by some to be essential for ALA synthesis. hemA and hemM were cloned separately into multi-copy plasmids. hemA was required for ALA biosynthesis in two ALA-deficient mutant strains. More ALA was produced by strains harboring a plasmid with both hemA and hemM than by those with hemA alone. These results suggest that hemA alone is required for ALA biosynthesis.;In order to study GTR, BL21(DE{dollar}\sb3),{dollar} was transformed with a pET14b-based vector, pWC01, harboring hemA in front of a T7 promoter. The transformed strain, WC1201, secreted ALA and porphyrins into the medium. Sonicates of the induction mixture exhibited strong ALA synthesis activity and increased GTS activity. This activity was measurable without the use of radioactive substrate showing that a substantial amount of GTR had formed. GTR was observable as a 46 kDa band by Brilliant Blue G staining of SDS-PAGE gels. Autoradiography on native gradient PAGE showed that GTR expressed in vivo by induction of WC1201 had a molecular weight of approximately 117 kDa. Gel filtration of the induced sonicate showed a peak of enzymatic activity at about 126 kDa. When pET14b- or pUC19-based plasmids harboring hemA and hemM were expressed in an in vitro transcription-translation system, native gradient PAGE showed a product with a molecular weight of approximately 175 kDa. When the 117 kDa and 175 kDa proteins were excised from their native gels respectively, and run on SDS PAGE, autoradiography showed bands at 46 kDa. These results suggest that the 117-126 kDa species may be a dimer of GTR associated with glutamyl-tRNA or a complex of GTR, GTS and glutamyl-tRNA.;E. coli RP523, a strain which cannot use ALA because there is a mutation in hemB, accumulates ALA under aerobic and anaerobic growth conditions. The amount of ALA accumulation in the medium of RP523 decreased when hemin was present in the medium, suggesting regulation by hemin. It was found that the activity of ALA synthesis in a cell-free extract of WC1201 was also inhibited by hemin or hemin arginate, suggesting feedback inhibition of GTR by hemin. RP523 accumulated less ALA when it was grown on glucose-supplemented rich medium than when it was grown without glucose.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
