Structural studies of type B proline utilization A (PutA) proteins

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] This thesis addresses the structural studies of the type B proline utilization A (PutA) proteins. Proline utilization A (PutA) catalyzes the oxidation of proline to glutamate. PutAs are divided into three types based on the domain architecture. Type A PutAs have a minimal catalytic core consisting of proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Type B PutAs have the minimal catalytic core and a C-terminal domain (CTD). Type C PutAs have an additional DNA binding domain apart from these three domains. Determining how PutAs vary structurally across species is key to understanding the ways these proteins use substrate channeling in proline catabolism. To address this, we determined the crystal structures of the type B PutAs from Sinorhizobium meliloti (SmPutA) and Corynebacterium freiburgense (CfPutA). The CTD of SmPutA and CfPutA consists of a β-flap fused to a non-catalytic Rossmann fold domain. The β-flap forms a lid over the substrate channeling cavity that is analogous to the dimerization flap in the type A PutAs. The SmPutA structure suggests that the CTD facilitates substrate channeling by helping to prevent diffusion of the intermediate into the bulk medium. High resolution SmPutA structures (1.5 Å - 1.8 Å) were used to determine the structural changes associated with ligand binding. Ligand binding to either active site closes the ion pair gate (Glu 225-Arg 488) that separates the PRODH active site and the substrate-channeling tunnel. Binding ligands in the P5CDH active site orders the PRODH active site which implies allosteric communication between the two active sites. Met452, Tyr485, Arg489, Glu492, and Asn493 undergo structural changes when the protein binds substrates and products. Unexpectedly, the ribityl chain of the FAD adopts two conformations in all E-P complexes. These structural changes suggest that the conformational changes are important in the substrate channeling mechanism of PutAs. Small-angle X-ray scattering (SAXS) was used to determine the oligomeric states of three PutAs namely CfPutA, Cellulomonas cellasea PutA, (CcPutA) and Corynebacterium jeikeium PutA (CjPutA), which are closely related in sequence. Determination of oligomeric states is important in understanding the different ways that PutA domains are arranged to form functional substrate channeling systems. Unexpectedly, SAXS analysis suggests these PutAs have different oligomeric states in solution. The SAXS data show that CfPutA is more than 90% monomeric and less than 10% dimeric in solution under the conditions used. In contrast, CjPutA is dimeric in solution. CcPutA appears to form dimers that are observed for other PutAs.