Biochemistry, Department of


First Advisor

Dr. Donald F. Becker

Date of this Version

Winter 12-2021


Before ProQuest publication:

Mao, Y. (2021). CHARACTERIZATION OF THE MULTIFUNCTIONAL ENZYME PROLINE UTILIZATION A [Unpublished Doctoral dissertation]. University of Nebraska-Lincoln.


A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy, Major: Biochemistry (Molecular Mechanism of Disease), Under the Supervision of Professor Donald F. Becker. Lincoln, Nebraska: December 2021

Copyright © 2021 Yizi Mao


Proline is a unique and important amino acid. Proline is a proteogenic amino acid and its metabolism is involved in many critical cellular functions. Therefore, proline metabolism is tightly regulated, and dysfunction of proline metabolism is related to human diseases. The first step of proline oxidization to ∆1-pyrroline-5-carboxylic acid (P5C) is catalyzed by proline dehydrogenase (PRODH). P5C is then non-enzymatically hydrolyzed to glutamate-γ-semialdehyde (GSA), which can be further oxidized to glutamate by P5C dehydrogenase (P5CDH/GSALDH). In Gram-negative bacteria, the PRODH and P5CDH enzymes are expressed as one polypeptide called proline utilization A (PutA). In some Gram-negative bacteria an additional ribbon-helix-helix (RHH) domain is at the N-terminus of PutA, thus imparting additional DNA-binding activity.

The PutA RHH domain recognizes putC, which is the intergenic DNA between the genes putP (encodes for a proline transporter) and putA (encodes for PutA). Thus, the trifunctional PutAs regulate their own gene expression and proline transport by sensing cellular proline levels. A linker region connecting the DNA binding domain and the PRODH domain is believed to be important for PutA to switch function from DNA binding to membrane binding. A detailed study of the linker region in PutA from Escherchia coli is presented in Chapter 2.

Two natural thiol-containing proline analogs, the thiazolidine-2-carboxylic acid (T2C) and thiazolidine-4-carboxylic acid (T4C), have been reported to have beneficial and toxic effects in different organisms. In Chapter 3, T2C and T4C are tested as substrates using the bifunctional PutA enzyme from Sinorhizobium meliloti (SmPutA) as model for proline catabolic enzymes. X-ray crystal structures of SmPutA in complex with thioproline analogs are available, making SmPutA ideal for enzymatic characterization with thioprolines.

The PRODH and P5CDH/GSALDH domains are physically close to each other in bifunctional PutAs, and tunnels have been found connecting both active sites, allowing direct translocation of P5C/GSA from PRODH to P5CDH/GSALDH. Chapter 4 presents kinetic characterization of SmPutA wild-type and site-specific mutants, including a tunnel blocking mutant, and two monofunctional SmPutA mutants that lack either PRODH or P5CDH/GSALDH activity.

Advisor: Donald F. Becker

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