Authors:

Lucas O Johnson

Date Created:

Not specified

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About Paper:

Cyclic tetrapeptides (CTPs) are part of a diverse class of natural products that are especially promising in clinical applications. This is due to their broad range of biological activities and superior therapeutic properties, such as specificity and proteolytic stability, compared to their linear counterparts. While chemical methods exist to access CTPs, these methods often require the presence of certain amino acids in certain positions, thereby limiting their applicability. Because of these limitations, there has been great interest in a class of enzymes that have been shown to catalyze the head-to-tail cyclization of linear peptides, penicillin binding protein-type thioesterases (PBP-TEs). There has, however, been little insight gained into the properties of these PBP-TEs that determine their substrate scopes. This study is aimed at evaluating DNA shuffling mutants of two known PBP-TEs with differing substrate scopes, WP516 and Ulm16, and site-directed mutagenesis of Ulm16. DNA shuffling was completed between the genes for WP516 and Ulm16 and active mutants were expressed, purified, and tested with several peptide substrates. In Ulm16, three residues were selectively mutated to more closely mimic WP516. This Ulm16 triple-mutant was assayed with several linear peptides to assess the impact that each mutation has on the activity of Ulm16. From these mutants and their activities, we aim to pinpoint the residues and characteristics of PBP-TEs that are important in determining substrate scope, guiding future enzyme engineering efforts toward a broadly useful biocatalyst. Keywords: [no keywords provided]

Source:

Purdue University / 2025

Topics:

No topics listed

Co-authors:

Lucas O Johnson