Structures of the PutA peripheral membrane flavoenzyme reveal a dynamic substrate-channeling tunnel and the quinone-binding site

Abstract

Proline utilization A (PutA) proteins are bifunctional peripheral membrane flavoenzymes that catalyze the oxidation of L-proline to L-glutamate by the sequential activities of proline dehydrogenase and aldehyde dehydrogenase domains. Located at the inner membrane of Gram-negative bacteria, PutAs play a major role in energy metabolism by coupling the oxidation of proline imported from the environment to the reduction of membrane-associated quinones. Here, we report seven crystal structures of the 1,004-residue PutA from Geobacter sulfurreducens, along with determination of the protein oligomeric state by small-angle X-ray scattering and kinetic characterization of substrate channeling and quinone reduction. The structures reveal an elaborate and dynamic tunnel system featuring a 75-Å-long tunnel that links the two active sites and six smaller tunnels that connect the main tunnel to the bulk medium. The locations of these tunnels and their responses to ligand binding and flavin reduction suggest hypotheses about how proline, water, and quinones enter the tunnel system and where L-glutamate exits. Kinetic measurements show that glutamate production from proline occurs without a lag phase, consistent with substrate channeling and implying that the observed tunnel is functionally relevant. Furthermore, the structure of reduced PutA complexed with menadione bisulfite reveals the elusive quinone-binding site. The benzoquinone binds within 4.0 Å of the flavin si face, consistent with direct electron transfer. The location of the quinone site implies that the concave surface of the PutA dimer approaches the membrane. Altogether, these results provide insight into how PutAs couple proline oxidation to quinone reduction.proline catabolism | X-ray crystallography | membrane association P roline catabolism is an important pathway in bioenergetics and has been implicated in tumor suppression (1), lifespan extension (2), production of fungal virulence factors (3), and bacterial virulence (4, 5). The pathway (Fig. 1A) comprises the flavoenzyme proline dehydrogenase (PRODH) and the aldehyde dehydrogenase (ALDH) superfamily member Δ 1 -pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH, also known as ALDH4A1). PRODH catalyzes the FAD-dependent oxidation of proline to P5C. P5CDH is a misnomer, as the enzyme catalyzes the oxidization of L-glutamate-γ-semialdehyde (GSA), which is the hydrolysis product of P5C. The electrons abstracted from proline by PRODH flow into the electron transport chain whereas the carbon skeleton of L-proline ultimately enters the citric acid cycle via α-ketoglutarate.Curiously, in Gram-negative bacteria, PRODH and P5CDH are combined into a single polypeptide, which is known as proline utilization A (PutA). Two functional classes of PutA exist: bifunctional and trifunctional (6). Bifunctional PutAs, which are the focus of this work, are peripheral membrane-associated enzymes that exhibit both PRODH and P5CDH catalytic activities. Localization of PutA at the inner bacterial membrane facilitates the efficient...

First Evidence for Substrate Channeling between Proline Catabolic Enzymes

“…The observed lag time is greater than that observed for PutA. The lag times reported for PutA enzymes thus far range from no apparent lag to 23 s for E. coli PutA (8,9,11). The longer lag time of the TtPRODH- (50).…”

“…Crystal structures of PutA enzymes from Bradyrhizobium japonicum (8) and Geobacter sulfurreducens (9) revealed that the two active sites are separated by a linear distance of 40 -50 Å and connected by a curved tunnel that traverses ϳ70 Å. Kinetic data for these enzymes are consistent with a substrate channeling mechanism (8,9).…”

“…The x-ray crystal structures of T. thermophilus PRODH (TtPRODH) and P5CDH (TtP5CDH) have been solved providing a solid framework for investigating substrate channeling with these enzymes (27,28). TtPRODH has a (␤␣) 8 barrel structure which is conserved in the PRODH domain of PutA (8,9,29). TtP5CDH shares the fundamental aldehyde dehydrogenase (ALDH) dimeric structure of the ALDH superfamily (28), which is found in PutA and P5CDH from eukaryotes (30,31).…”

“…In these assays, the effects of inactive TtP5CDH and TtPRODH mutants on the overall coupled reaction rate were determined. The inactive TtPRODH mutant R288M/R289M was generated by replacing two catalytic arginine residues (Arg-288 and Arg-289) that are essential in PRODH and PutA for proline binding via ionic interactions with the carboxylate moiety of proline (9,30,41,42). Fig.…”

Mini Review Identifying human disease genes: advances in molecular genetics and computational approaches

“…This method requires prior knowledge of a particular gene in the pathogenesis of the disorder. The gene and surrounding DNA is analyzed through case-control association studies for comparison among people with and without disease to figure out alterations specific to people having the disease phenotype (Singh et al, 2014). Similarly, allele specific methods and direct sequencing of entire candidate region in both cases and controls are also exploited.…”