Author ORCID Identifier

0000-0001-5848-4675

Date of Award

12-13-2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Giovanni Gadda

Second Advisor

Donald Hamelberg

Third Advisor

Irene T. Weber

Abstract

Pseudomonas aeruginosa has a two-enzyme D-arginine conversion system that enables it to utilize D-arginine as a nutrient. The coupled-enzyme system consists of D-arginine dehydrogenase (PaDADH) and L-arginine dehydrogenase (PaLADH). PaDADH has been characterized structurally and mechanistically and established as an FAD-dependent enzyme that catalyzes the oxidation of D-arginine to iminoarginine, which can be non-enzymatically hydrolyzed to ketoarginine and ammonia.

A tyrosine 249 to phenylalanine variant of PaDADH (PaDADH-Y249F) was expressed and purified as a mixture of two cofactors, one protein population with regular FAD and one with a green-colored modified FAD. The chemically modified green FAD was investigated through various spectroscopic techniques. The yellow and green protein fractions were investigated structurally to uncover any clues about the modification. Molecular Dynamics and QM/MM were also performed with the wild-type and the variant enzymes to investigate the contribution of protein dynamics and flavin electronics into making the 6-hydroxylation reaction possible in the variant enzyme.

PaDADH-Y249F was also expressed and crystallized in the presence of D-arginine. The structure of this incubated enzyme was similar in overall topology to the wild-type enzyme, except for ambiguous electron density around the N5 and C6 atoms of the isoalloxazine moiety. The FAD modified at the N5 position was present in the variant enzyme and the green modified FAD. The presence of two modified flavin cofactors in a single crystal structure is unusual. The second modified FAD was characterized via accurate mass analysis and investigated spectroscopically.

While a lot is known about PaDADH, PaLADH has yet to be characterized kinetically. PaLADH is the second enzyme in the coupled-enzyme D-arginine conversion system and catalyzes the reaction of ketoarginine and ammonia to L-arginine using NAD(P)H. PaLADH can also catalyze the reverse reaction converting L-arginine to ketoarginine and ammonia using NAD(P)+ and was characterized using a stopped-flow spectrophotometer. The forward reaction of PaLADH was investigated as the product of the coupled-enzyme reaction by providing the nicotinamide substrate and D-arginine for PaDADH, with PaLADH also being present in the same reaction mix. We also hypothesize that PaLADH and PaDADH might form a protein complex for converting D-arginine to L-arginine, and the complex was probed using analytical ultracentrifugation and size-exclusion chromatography.

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