Author ORCID Identifier

https://orcid.org/0000-0002-7763-0380

Date of Award

Fall 12-13-2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Gioavanni Gadda

Second Advisor

Dabney W. Dixon

Third Advisor

Donald Hamelberg

Abstract

Enzymes are highly flexible biocatalysts in biological organisms. X-ray crystallography, nuclear magnetic resonance studies, and computational simulations data indicate that enzymes are dynamic networks of amino acid residues and undergo multiple conformational changes during catalysis. Understanding the interconnection between enzyme dynamics and catalysis or catalysis-linked dynamics is a broad subject of interest for modern scientists. However, the role of enzyme dynamics in catalysis is still not fully understood. This dissertation used D-arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH) as a model enzyme to investigate enzyme dynamics and catalysis.

PaDADH is part of a two-enzyme system that catalyzes the irreversible conversion of D-arginine to L-arginine. The three-dimensional structure of PaDADH shows four loops (L1-L4) that are part of the active site. Loop L1 has two different peptidyl regions including residues 45-47 located at the FAD-binding domain and residues 50-56 positioned at the entrance of the active site. In the two peptidyl regions of loop L1, only the S45, A46 and Y53 residues adopt major conformational changes corresponding to the open (ligand-free) and closed (product-bound) conformations. Loop L2 is positioned at the entrance of the active site pocket. In loop L2, only E246 points toward the active site and interacts with the product and forms an hydrogen-bond with Y53 residue. Loop L3 is on the surface and interacts with the rigid portion of loop L1. Loop L4 is buried inside the protein matrix and spans over the isoalloxazine and the ribityl moieties of the flavin cofactor.

Site-directed mutagenesis was used to target key amino acid residues in these loops. The engineered variant enzymes were characterized using computational simulations and experimental techniques. The data showed that the peptidyl regions of loop L1 are interconnected and that the Y53 residue acts as a gate to control the active site access. The integrity of loop L4 is important to maintaining the overall conformation of PaDADH and to accommodate of the enzyme-substrate and product complexes. Taken together the data indicate that the dynamics of the active site loops play a significant role in the ability of PaDADH to bind its substrate and perform catalysis

DOI

https://doi.org/10.57709/26800779

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