Studies of lipoic acid and biotin metabolism in bacteria and higher eukaryotes

Cao, Xinyun

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https://hdl.handle.net/2142/101113 Copy

Description

Title

Studies of lipoic acid and biotin metabolism in bacteria and higher eukaryotes

Author(s)

Cao, Xinyun

Issue Date

2018-02-02

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

Cronan, John E.

Doctoral Committee Chair(s)

Cronan, John E.

Committee Member(s)

• Gennis, Robert B.
• Nair, Satish K.
• Jin, Hong

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
• biotin
• lipoate protein ligase
• methyl esterase
• octanoyl transferase
• circular permutation
• LplA
• BioH
• LipM
• glycine cleavage
• enzyme moonlighting
• aerobic metabolism
• C1 metabolism

Abstract

My PhD research focus has been the study of lipoic acid and biotin metabolism in several bacteria. Lipoic acid and biotin are essential enzyme cofactors found in all three domains of life. Both molecules function only when covalently attached to key metabolic enzymes, 2-oxoacid dehygrogenase (OADH) and glycine cleavage H protein (GcvH). There they act as “swinging arms” that transfer intermediates between two active sites of key metabolic enzymes by covalent substrate channeling. Escherichia coli and Bacillus subtilis are the two organisms with well characterized but quite distinct models for lipoic acid and biotin metabolism. In this thesis I made further studies about lipoic acid synthesis genes in other organisms and resolved remaining questions in biotin synthesis. I also discovered novel functions and mechanisms of proteins involved in lipoic acid metabolic pathway by bacteria genetics and biochemical assays. In the second chapter I investigated the lipoate-protein liagase (LplA) from Streptomyces coelicolor. LplA enzymes function to scavenge lipoic acid from the environment and attach the coenzyme to its cognate proteins, which are generally the E2 components of the 2-oxoacid dehydrogenases. The LplA reaction is catalyzed in two steps: (1) LplA activates lipoate to lipoyl-AMP, (2) LplA transfers the lipoyl-moiety from lipoyl-AMP to the cognate protein. The S. coelicolor genome encodes only a single putative lipoate ligase and this protein had very low sequence identity to the well characterized E. coli LplA (<25%) and has the domain order reversed relative to the E. coli protein. I tested the activity of S. coelicolor LplA both by in vivo complementation of an E. coli ligase-deficient strain and by in vitro assays. Moreover, when I rearranged S. coelicolor LplA into one that mimicked the arrangement found in the canonical lipoate ligases, the enzyme retained complementation activity. Finally, when the two domains were separated into two proteins, both domain-containing proteins were required for complementation and catalysis of the overall ligase reaction in vitro. However, only the large domain-containing protein was required for transfer of lipoate from the lipoyl-AMP intermediate to the acceptor proteins, whereas both domain-containing proteins were required to form lipoyl-AMP. In the third chapter, I unraveled the puzzle of different BioH proteins-one of the enzymes involved in biotin biosynthesis. The late steps of biotin synthesis, which are responsible for assembly of the rings, are conserved among different organisms and were well described biochemically years ago. These steps comprise a cluster of four genes, bioABFD. In contrast, the early steps of biotin synthesis, assembly of the valeric acid side-chain of biotin were determineded recently in E. coli and only two genes, bioC and bioH are required for this. The BioC methyltransferase disguises the substrate to allow entry into the fatty acid synthesis pathway by methylating the free carboxyl of malonyl-acyl carrier protein (ACP). When chain has been elongated to seven carbons, the disguise is removed the BioH esterase. Although the biotin synthesis pathway has been well demonstrated, there remained a puzzle. In E. coli, the bioH (“freestanding” bioH) gene is located at a site well removed from the biotin synthetic operon and is not regulated by the BirA repressor whereas in other proteobacteria (e.g., the pseudomonads) the bioH gene (“operon-encoded” bioH) is found in an apparent biotin operon and in general is located immediately upstream of bioC. Moreover, E. coli BioH is a rather promiscuous hydrolase and has much higher enzymatic activity compared to the other biotin synthetic enzymes. In this chapter, I compared and contrasted the two different kinds of bioH: “freestanding” bioH from E. coli and “operon-encoded” bioH from P. aeruginosa in terms of their enzymatic activity, substrate specificity, transcription and translation levels. I found both BioHs have similar high enzymatic activity and broad range of substrate specificity. However, the “freestanding” bioH is transcribed and translated at lower levels than the “operon-encoded” bioH. In the fourth chapter we investigated a remarkable case in which the GcvH protein’s “moonlighting” function (i.e., development of a new function while retaining the original function) is found throughout evolution in the absence of selection. The B. subtilis lipoic acid synthesis pathway is primordial due to the fact that GcvH is the only substrate for lipoate assembly. Hence in B. subtilis, lipoic acid-requiring 2-oxoacid dehydrogenase (OADH) proteins acquire the cofactor only by transfer from lipoylated GcvH. The E. coli pathway where lipoate is directly assembled on both its GcvH and OADH proteins, is proposed to arise later. Since roughly two billion years separate the divergence of these two bacteria (E. coli and B. subtilis), it is surprising that E. coli GcvH functionally substitutes for the B. subtilis protein in lipoyl transfer. I tested several known and putative GcvHs from other bacteria and eukaryotes by in vivo complementation and in vitro enzymatic assays and found they could also substitute for B. subtilis GcvH in OADH modification. In the fifth chapter, I summarize my findings and give my outlooks especially for avenues of lipoic acid metabolism studies. Science is so beautiful and there are numerous interesting questions that haven’t been fully studied yet. I hope my thesis will provide insight and thoughts for people in the future study of lipoic acid and biotin related problems.

Graduation Semester

2018-05

Type of Resource

text

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Copyright 2018 Xinyun Cao

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