Homologation of L-Phe and L-Tyr in Natural Product Biosynthesis: Characterization of Enzyme HphCD and HphA Mutations

Date

2023-04

Authors

Lang, Rebecca

Journal Title

Journal ISSN

Volume Title

Publisher

Augusta University

Abstract

The discovery and development of drugs is extremely important in the medical field, with natural products (NPs) being especially useful for drug modification. One of the enzymatic families that produce extremely diverse NPs, called nonribosomal peptide synthetases (NRPSs), may be used in bioengineering methods that modify drug production pathways. The amino acid building blocks that are employed in NRPSs may also be modified by various pathways prior to entering NRPSs. One such pathway that modifies these building blocks is the homologation pathway, which is being examined in this project. The homologation pathway adds a carbon and two hydrogens (a methylene group) onto an amino acid side chain, and it may be used to diversify the number of products in a synthetic pathway. This project had two aims: to characterize one of the enzymes, named HphCD involved in the homologation pathway, and to analyze mutations of another enzyme in the pathway, HphA.

The production of enzyme HphCD was carried out via overexpression of the gene that codes for it, hphCD, after it had been cloned into the proper expression plasmid (pET-28a or pMAL-c6T) in an Escherichia coli (E. coli) strain. 1, 2 Following the overexpression of HphCD, the enzyme was supposed to be characterized via enzymatic assays, which would have facilitated the determination of the enzyme’s activity and substrate specificity. However, the enzyme was not properly obtained in a soluble form even after various methods were attempted, and therefore HphCD could not be characterized.

The second part of the project generated two mutants of wild-type HphA: H218A and H220A, the residues proposed to form a Zn 2+ binding site. These mutants were cloned into pET-28a plasmids and overexpressed to obtain the mutated enzymes. Following overexpression, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to characterize 5 the expression level, solubility in an aqueous buffer, and purity of the protein. Then, since both mutated enzymes were obtained via overexpression and purification, enzymatic assays were performed on each mutant. It was found that H218A disrupted the normal function of the enzyme, while H220A did not. More assays will need to be carried out to determine the role of the Zn 2+ binding site of HphA.

Description

The file you are attempting to access is restricted to Augusta University. Please login using your JagNet iD and password.

Keywords

Citation

DOI