The Structure of the Proline Utilization A Proline Dehydrogenase Domain Inactivated by N-Propargylglycine Provides Insight into Conformational Changes Induced by Substrate Binding and Flavin Reduction^,

Abstract

Proline utilization A (PutA) from Escherichia coli is a flavoprotein that has mutually exclusive roles as a transcriptional repressor of the put regulon and a membrane-associated enzyme that catalyzes the oxidation of proline to glutamate. Previous studies have shown that the binding of proline in the proline dehydrogenase (PRODH) active site and subsequent reduction of the FAD trigger global conformational changes that enhance PutA-membrane affinity. These events cause PutA to switch from its repressor to enzymatic role, but the mechanism by which this signal is propagated from the active site to the distal membrane-binding domain is largely unknown. Here, it is shown that N-propargylglycine irreversibly inactivates PutA by covalently linking the flavin N(5) atom to the ε-amino of Lys329. Furthermore, inactivation locks PutA into a conformation that may mimic the proline reduced, membrane-associated form. The 2.15 Å resolution structure of the inactivated PRODH domain suggests that the initial events involved in broadcasting the reduced flavin state to the distal membrane binding domain include major reorganization of the flavin ribityl chain, severe (35 degree) butterfly bending of the isoalloxazine ring, and disruption of an electrostatic network involving the flavin N(5), Arg431, and Asp370. The structure also provides information about conformational changes associated with substrate binding. This analysis suggests that the active site is incompletely assembled in the absence of the substrate, and the binding of proline draws together conserved residues in helix 8 and the β1-αl loop to complete the active site.

Flavin Redox Switching of Protein Functions

“…not observed in the L-THFA bound structure (69,83). Another significant conformational change in the FAD cofactor was the formation of a new hydrogen bond between the 2 0 -OH ribityl group and the FAD N(1) position (69,83).…”

“…In the first reduced structure, the PRODH domain crystal form was reduced with dithionite (83). In the second structure, N-propargylglycine (PPG) was used to covalently modify the FAD (69). PPG irreversibly inactivates EcPutA by generating a three carbon link between active site residue K329 and the FAD N(5) atom.…”

“…In addition, limited proteolysis of PPG-inactivated EcPutA indicated that the inactivated enzyme is locked into a conformation that resembles prolinereduced EcPutA. Thus, it was proposed that PPG inactivated EcPutA mimics the proline-reduced, membrane-bound form of PutA (69).…”

“…Another significant conformational change in the FAD cofactor was the formation of a new hydrogen bond between the 2 0 -OH ribityl group and the FAD N(1) position (69,83). In crystal structures of oxidized PRODH, the 2 0 -OH ribityl group of FAD hydrogen bonds to Arg556, thus, reduction of the FAD causes the 2 0 -OH group to rotate 90 o (83) (Fig.…”

“…These proteins were chosen because recent biochemical and structural studies of these flavin switch proteins have provided novel details into how cofactor reduction=modification leads to the propagation of conformational and functional changes (50,69,83,90). Two of the proteins, VVD and NifL, contain a flavin-binding Per Arnt Sim (PAS) domain (64,66).…”