Discovery of the Membrane Binding Domain in Trifunctional Proline Utilization A

Abstract

Escherichia coli proline utilization A (EcPutA) is the archetype of trifunctional PutA flavoproteins, which function both as regulators of the proline utilization operon and bifunctional enzymes that catalyze the four-electron oxidation of proline to glutamate. EcPutA shifts from a self-regulating transcriptional repressor to a bifunctional enzyme in a process known as functional switching. The flavin redox state dictates the function of EcPutA. Upon proline oxidation, the flavin becomes reduced, triggering a conformational change that causes EcPutA to dissociate from the put regulon and bind to the cellular membrane. Major structure/function domains of EcPutA have been characterized, including the DNA-binding domain, proline dehydrogenase (PRODH) and L-glutamate-γ-semialdehyde dehydrogenase catalytic domains, and an aldehyde dehydrogenase superfamily fold domain. Still lacking is an understanding of the membrane-binding domain, which is essential for EcPutA catalytic turnover and functional switching. Here, we provide evidence for a conserved C-terminal motif (CCM) in EcPutA having a critical role in membrane binding. Deletion of the CCM or replacement of hydrophobic residues with negatively charged residues within the CCM impairs EcPutA functional and physical membrane association. Furthermore, cell-based transcription assays and limited proteolysis indicate that the CCM is essential for functional switching. Using fluorescence resonance energy transfer involving dansyl-labeled liposomes, residues in the α-domain are also implicated in membrane binding. Taken together, these experiments suggest that the CCM and α-domain converge to form a membrane-binding interface near the PRODH domain. The discovery of the membrane-binding region will assist efforts to define flavin redox signaling pathways responsible for EcPutA functional switching.

Exploring the Potential Role of Moonlighting Function of the Surface-Associated Proteins From Mycobacterium bovis BCG Moreau and Pasteur by Comparative Proteomic

“…The moonlighting domain hit is PutA, a trifunctional protein in bacteria: transcriptional regulator, proline dehydrogenase and pyrroline-5- carboxylate dehydrogenase (data not shown). Christgen et al (180) discovered a membrane binding region on the PutA domain from Escherichia coli AldC that explains the PutA functional switch from self-regulating transcriptional repressor to membrane binding domain. Our results indicate that AldC is a surface-associated protein from M. bovis BCG Moreau (spot 171 of Figure 1A) not found in the culture filtrate of BCG Moreau (Table S2).…”

Proline utilization A controls bacterial pathogenicity by sensing its substrate and cofactors

“…PutA was previously demonstrated to act as a bifunctional enzyme that converts proline to glutamate and to be a negative regulator of the proline utilization operon, while proline switches PutA from a self-regulating transcriptional repressor to an enzymatic function in some bacterial species 9 . To investigate whether L-proline plays a role in the regulatory activity of PutA in R. solanacearum , we first tested the effect of L-proline on EPS biosynthesis.…”

“…Proline is usually used as an important source of carbon and nitrogen and plays a vital role in many biological processes, such as cellular biosynthesis, cell growth, oxidative stress responses, osmotic stress responses, and redox signalling 1 – 4 . It can be oxidized to glutamic acid via the coordinated activity of proline dehydrogenase (PRODH), L-glutamate-γ-semialdehyde dehydrogenase (GSALDH, also named P5C dehydrogenase and ALDH4A1) and cofactors 4 – 9 . The enzyme that catalyses the oxidation of L-proline to L-glutamic acid is called proline utilization A (PutA) in many bacteria 7 , 9 – 11 .…”

“…It can be oxidized to glutamic acid via the coordinated activity of proline dehydrogenase (PRODH), L-glutamate-γ-semialdehyde dehydrogenase (GSALDH, also named P5C dehydrogenase and ALDH4A1) and cofactors 4 – 9 . The enzyme that catalyses the oxidation of L-proline to L-glutamic acid is called proline utilization A (PutA) in many bacteria 7 , 9 – 11 . The domain architecture of PutA proteins indicates that they are mainly bifunctional enzymes that combine PRODH and GSALDH activity and trifunctional enzymes with an additional DNA-binding domain functioning as a transcriptional repressor of proline utilization genes 7 , 11 .…”

Functional Impact of the N-terminal Arm of Proline Dehydrogenase from Thermus thermophilus

“…After P5C hydrolysis, the resulting glutamic semialdehyde (GSA) is oxidized to glutamate through the action of Δ 1 -pyrroline-5-carboxylate dehydrogenase (P5CDH; EC 1.2.1.88) ( Scheme 1 ). ProDH and P5CDH exist as separate monofunctional enzymes in eukaryotes and some bacteria, but are fused into a bifunctional [ 1 , 2 ] or trifunctional [ 3 ] enzyme in other bacteria. In these multi-functional enzymes, called proline utilization A (PutA), the C -terminus of ProDH is fused to P5CDH, allowing for the channeling of the P5C/GSA intermediate between the enzymes [ 4 , 5 , 6 , 7 , 8 , 9 ].…”