Lipoic acid assembly on 2-oxoacid dehydrogenases in E.coli

El Haj Hassan, Bachar H.

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Description

Title

Lipoic acid assembly on 2-oxoacid dehydrogenases in E.coli

Author(s)

El Haj Hassan, Bachar H.

Issue Date

2011-05-25T14:24:51Z

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

Cronan, John E.

Doctoral Committee Chair(s)

Cronan, John E.

Committee Member(s)

• Sligar, Stephen G.
• Gennis, Robert B.
• Gerlt, John A.

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Lipoic acid
• pyruvate dehydrogenase
• 2-oxoglutarate dehydrogenase
• LipB
• LipA
• LplA
• GroEL

Abstract

Lipoic acid is a covalently attached cofactor essential for the activity of 2-oxoacid dehydrogenases and the glycine cleavage system. In the absence of lipoic acid modification the dehydrogenases are inactive and aerobic metabolism is blocked. In Escherichia coli two pathways for the attachment of lipoic acid exist, a de novo biosynthetic pathway dependent on the activities of the LipB and LipA proteins and a lipoic acid scavenging pathway catalyzed by the LplA protein. LipB is responsible for octanoylation of the E2 components of 2-oxoacid dehydrogenases to provide the substrates of LipA, a SAM radical enzyme that inserts two sulfur atoms into the octanoyl moiety to give the active lipoylated dehydrogenase complexes. We report that the intact pyruvate and 2-oxoglutarate dehydrogenase complexes specifically copurify with both LipB and LipA. Proteomic, genetic and dehydrogenase activity data indicate that all of the 2-oxoacid dehydrogenase components are present. In contrast LplA, the lipoate protein ligase enzyme of lipoate salvage, shows no interaction with the 2-oxoacid dehydrogenases. The interaction is specific to the dehydrogenases in that the third lipoic acid requiring enzyme of E. coli, the glycine cleavage system H protein, does not copurify with either LipA or LipB. Studies of LipB interaction with engineered variants of the E2 subunit of 2-oxoglutarate dehydrogenase indicate that binding sites for LipB reside both in the lipoyl domain and catalytic core sequences. We also report that LipB forms a very tight, albeit noncovalent, complex with acyl carrier protein. These results indicate that lipoic acid is not only assembled on the dehydrogenase lipoyl domains, but that the enzymes that catalyze the assembly are also present “on site”. We have also investigated a possible alternative route for lipoic acid biosynthesis in which Lipoic acid is synthesized on ACP and transferred by LipB to unmodified lipoyl domains. Since LipB was demonstrated to proceed through an acyl-enzyme intermediate, we attempted to isolate an acylated peptide from LipB, and isolated the unmodified peptide containing the active site cysteine C169. However no acylated peptide was found. We demonstrate biochemically that the octanoylated peptide is cleaved in the trypsin digestion reaction possibly by attack by the two other cysteine residues (C137 and C147). Chemical transfer of octanoyl from octanoyl-imidazole to the peptide was also unsuccessful suggesting an intrinsic instability of the peptidyl thioester bond. Consequently we resorted to study the intact LipB proteins by total hydrolysis and GC/MS and found that the enzyme-bound acyl intermediate is octanoic acid rather than lipoic acid. Octanoyl-ACP was not a substrate for LipA in vivo thus providing further evidence that LipB is an Octanoyl-[Acyl Carrier Protein]:Protein N-Octanoyltransferase and not a Lipoyl transferase in vivo.

Graduation Semester

2011-05

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Copyright and License Information

© 2011 Bachar El Haj Hassan

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