Studies on lipoic acid metabolism

Hermes, Fatemah

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https://hdl.handle.net/2142/72926 Copy

Description

Title

Studies on lipoic acid metabolism

Author(s)

Hermes, Fatemah

Issue Date

2015-01-21

Director of Research (if dissertation) or Advisor (if thesis)

Cronan, John E.

Doctoral Committee Chair(s)

Cronan, John E.

Committee Member(s)

• Farrand, Stephen K.
• Imlay, James A.
• Orlean, Peter A.

Department of Study

Microbiology

Discipline

Microbiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• lipoic acid
• tricarboxylic acid (TCA)

Abstract

The covalent attachment of lipoic acid to the lipoyl domains (LDs) of the central metabolism enzymes pyruvate dehydrogenase (PDH) and oxoglutarate dehydrogenase (OGDH) is essential for their activation and thus for respiratory growth in the bacterium Escherichia coli and the yeast Saccharomyces cerevisiae. A third lipoic acid-dependent enzyme system, the glycine cleavage system (GCV), is required for utilization of glycine as a nitrogen source. In E. coli, lipoic acid is assembled on the LDs from the eight-carbon fatty acid, octanoate, in two steps. First, an octanoyltransferase (LipB) transfers an octanoyl moiety from the acyl carrier protein (ACP) of fatty acid biosynthesis to the LDs. A thioester-bound acyl-enzyme intermediate is formed in the process. Then lipoyl synthase (LipA) catalyzes replacement of single hydrogen atoms at carbons 6 and 8 with sulfur atoms using radical SAM chemistry. Alternatively, either exogenous lipoic acid or octanoate can be directly attached to the LDs by lipoate-protein ligase (LplA) via an acyl–AMP intermediate. E. coli strains containing null mutations in lipB are auxotrophic for either lipoic acid (or octanoate), or acetate plus succinate which respectively bypass the PDH- and OGDH-catalyzed steps required for aerobic growth on glucose minimal media. Spontaneously-arising mutant strains that retained the lipB mutation, yet did not require supplementation for aerobic growth were isolated. Initial characterization distinguished two types of suppressor strains. In chapter 2 I describe one type in which suppression was caused by single missense mutations within the coding sequence of the lplA gene. The LplA proteins encoded by the mutant genes had reduced Km values for free octanoate, which was detected in the cytoplasm at a concentration of about 28.2 μM, well above the Km values for the mutant LplA proteins. Thus in these suppressor strains, the mutant LplA proteins utilize the cytoplasmic octanoate pool to activate PDH and OGDH enabling growth. In the second type of lipB suppressor strains, the causative mutation was a stop codon in the sdhB gene, which encodes a subunit of succinate dehydrogenase (SDH). In chapter 3, these lipB sdhB strains are further characterized. I show that these strains contain active PDH and require a functional lplA gene. Succinate in this strain is produced by three enzymes, any one of which will suffice in the absence of SDH. These three enzymes are: trace levels of OGDH, the isocitrate lyase of the glyoxylate shunt, and aspartate oxidase, the enzyme catalyzing the first step of nicotinamide biosynthesis. In chapter 4, I characterize the lipoate-protein ligase of the yeast S. cerevisiae. The E. coli LplA was the first lipoate-protein ligase (Lpl) to be characterized. It catalyzes two partial reactions: activation of the acyl chain by formation of acyl-AMP, followed by transfer of the acyl chain to the LDs. It turns out that there is a surprising diversity within the Lpl family of enzymes, several of which catalyze reactions other than ligation reactions. For example, the Bacillus subtilis Lpl homologue LipM is an octanoyltransferase that transfers the octanoyl moiety from octanoyl-ACP specifically to GCV. Another B. subtilis Lpl homologue, LipL, transfers octanoate from octanoyl-GCV to other LDs in an amido-transfer reaction. In chapter 4, I report that the Lip3 Lpl homologue of S. cerevisiae has octanoyl-CoA:protein transferase activity, and discuss implications of this activity on the physiological role of Lip3 in lipoic acid synthesis.

Graduation Semester

2014-12

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http://hdl.handle.net/2142/72926

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Copyright 2014 Fatemah Hermes

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