Mutant Study of Sinorhizobium meliloti Proline Utilization A (PutA)

The purpose of this project is to purify and characterize the reaction kinetics of mutant versions the enzyme Proline Utilization A (PutA) in Sinorhizobium meliloti. The enzyme catalyzes the first step in proline metabolism. It has two active sites. The first is proline dehydrogenase (PRODH) which converts proline to pyrroline-5-carboxylate (P5C). The second is P5C dehydrogenase (P5CDH) which converts P5C to glutamate. Although many bacterial organisms have PutA, there are still significant interspecies variations, resulting in an entire family of PutA enzymes. The main difference is the length of the amino acid sequence. This affects the protein’s structure or its shape, and the protein’s kinetics or how it behaves in reactions. In order to have a complete understanding of proline metabolism, all the variations of PutA must be characterized both structurally and kinetically. The version of PutA found in S. meliloti (SmPutA) has been categorized structurally but not kinetically. This project aims to fill this gap in our knowledge of proline metabolism and PutA from S. meliloti