Studies of Endogenous Biosynthesis of Lipoic Acid Cofactor in Humans: Expression, Purification and Characterization of LIPT1 and LIPT2 Enzymes
Open Access
- Author:
- Mosley, Ilana
- Area of Honors:
- Veterinary and Biomedical Sciences
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Squire J Booker, Thesis Supervisor
Robert John Vansaun, Thesis Honors Advisor - Keywords:
- biochemistry
lipoic acid
malaria - Abstract:
- Studies of Endogenous Biosynthesis of Lipoic Acid Cofactor in Humans: Expression, Purification and Characterization of LIPT1 and LIPT2 Enzymes Ilana A. Mosley 1, Douglas Warui 1, Ph.D., and Squire Booker 1, Ph.D. The Pennsylvania State University, University Park, Pennsylvania 1 Lipoic acid is a cyclic disulfide that plays a role in intermediate metabolism. It exists predominantly in the form of its conjugate base, lipoate, under physiological conditions. In humans, lipoate is essential for five redox reactions, four 2-oxoacid dehydrogenases and the glycine cleavage system. In this study, two enzymes, LIPT1 and LIPT2, were investigated. LIPT2 transfers octanoic acid from ACP to GCSH and LIPT1 transfers lipoic acid from GCSH to DLST. GCSH catalyzes the degradation of glycine and mutations in the gene are linked to hyperglycemia. Mutations in DLST have been linked to the development of Alzheimer’s in elderly populations. DLST is also an essential part of the Kreb’s cycle, therefore mutations in the gene can lead to issues converting ADP to ATP. The primary goal of the study was to overexpress and purify the genes involved in the human lipoic acid biosynthesis pathway so that the activity of LIPT1 and LIPT2 could be observed in vitro. In order to characterize the activity of LIPT1 and LIPT2, both genes along with DLST, AaS, ACPS1, Ulp1, and ACP were cloned into dwpSUMO and pET28a plasmids using DH5a E. coli cells. Afterwards, the plasmids were transformed into BL21-DE3 E. coli cells, overexpressed, and purified. All of the genes of interest were successfully cloned into the dwpSUMO and pET28a plasmids and transformed into BL21-DE3 E. coli cells. AaS and Ulp1 were successfully overexpressed and purified. Preliminary results displayed separation between the standards for holo-ACP and acyl-ACP. However, the separation between the peaks corresponding to the holo- GCSH and lipoyl- GCSH was inadequate. Therefore, the HPLC method requires additional alterations before assays can be conducted. Future directions include overexpressing DLST, ACPS1, and ACP as well as monitoring the lipoic acid transfers by LIPT1 and LIPT2 via HPLC analysis.