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Investigation of structure-function relationships in the bifunctional PutA enzyme and the role of proline in modulating the redox environment

Navasona Krishnan, University of Nebraska - Lincoln

Abstract

Proline oxidation to glutamate is catalyzed by the flavoenzyme PutA in several microorganisms. In certain prokaryotes such as Escherichia coli, PutA is also a transcriptional repressor of the proline utilization (put) genes and thus is trifunctional. We have begun to assess differences between bifunctional enzymes from Bradyrhizobium japonicum (BjPutA) and Helicobacter hepaticus (HhPutA) and the trifunctional enzyme from Escherichia coli (EcPutA). The bifunctional enzymatic activity is shared among all PutA enzymes with interesting differences attributing to the new properties exhibited by BjPutA and HhPutA. A reduction potential (Em) value of -0.132 V (pH 7.5) was determined for the bound FAD/FADH2 couple in BjPutA that is significantly more negative (∼55 mV) than the Em for EcPutA-bound FAD. This situation thermodynamically limits proline reduction of the FAD cofactor in BjPutA. In the presence of phospholipids, reduction of BjPutA is stimulated; suggesting lipids influence the FAD redox environment. Limited proteolysis of BjPutA under reducing conditions shows FAD reduction does not influence BjPutA conformation suggesting that the redox dependent regulation observed with EcPutA may be limited to trifunctional PutA homologues. The possibility of substrate channeling in proline catabolism was tested in 2 BjPutA. Steady-state and stopped-flow kinetic measurements provide clear evidence for a channeling mechanism in BjPutA. ^ The characterization of HhPutA showed that it exhibits about 100-fold higher oxidase activity than EcPutA. The significance of increased oxygen reactivity in HhPutA was studied by oxidative stress studies in E. coli. To understand the physiological relevance of proline metabolism, a mutant strain of H. hepaticus was generated by deleting the putA gene. From a mouse colonization studies it is apparent that PutA is imperative in causing increased inflammation and contributes to the pathology of H. hepaticus infection. ^ Proline metabolism has been associated with a number of different ecological niches, with proline having multifaceted roles in protein chaperoning, abiotic stress protection, and energy utilization. A less understood property of proline is its ability to scavenge free radicals in mammals. The potential of proline to suppress ROS levels and apoptosis in mammalian cells was studied. Proline was effective in rescuing cells from H2O2 mediated apoptosis. In cells exposed to oxidative stress, proline biosynthesis was upregulated. The proline catabolic enzyme (PRODH) was purified and showed to generate ROS as a consequence of substrate reduction of the FAD cofactor. These results suggest that proline uniquely impacts the intracellular redox environment by opposing mechanisms. ^

Subject Area

Chemistry, Biochemistry

Recommended Citation

Krishnan, Navasona, "Investigation of structure-function relationships in the bifunctional PutA enzyme and the role of proline in modulating the redox environment" (2008). ETD collection for University of Nebraska - Lincoln. AAI3290780.
http://digitalcommons.unl.edu/dissertations/AAI3290780

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