Donald F. Becker

Proline Mechanisms of Stress Survival

SIGNIFICANCE The imino acid proline is utilized by different organisms to offset cellular imbalances caused by environmental stress. The wide use in nature of proline as a stress adaptor molecule indicates that proline has a fundamental biological role in stress response. Understanding the mechanisms by which proline enhances abiotic/biotic stress response will facilitate agricultural crop research and improve human health. RECENT ADVANCES It is now recognized that proline metabolism propels cellular signaling processes that promote cellular apoptosis or survival. Studies have shown that proline metabolism influences signaling pathways by increasing reactive oxygen species (ROS) formation in the mitochondria via the electron transport chain. Enhanced ROS production due to proline metabolism has been implicated in the hypersensitive response in plants, lifespan extension in worms, and apoptosis, tumor suppression, and cell survival in animals. CRITICAL ISSUES The ability of proline to influence disparate cellular outcomes may be governed by ROS levels generated in the mitochondria. Defining the threshold at which proline metabolic enzyme expression switches from inducing survival pathways to cellular apoptosis would provide molecular insights into cellular redox regulation by proline. Are ROS the only mediators of proline metabolic signaling or are other factors involved? FUTURE DIRECTIONS New evidence suggests that proline biosynthesis enzymes interact with redox proteins such as thioredoxin. An important future pursuit will be to identify other interacting partners of proline metabolic enzymes to uncover novel regulatory and signaling networks of cellular stress response.

Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus.

Proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) catalyze the two-step oxidation of proline to glutamate. They are distinct monofunctional enzymes in all eukaryotes and some bacteria but are fused into bifunctional enzymes known as proline utilization A (PutA) in other bacteria. Here we report the first structure and biochemical data for a monofunctional PRODH. The 2.0-Å resolution structure of Thermus thermophilus PRODH reveals a distorted (βα)8 barrel catalytic core domain and a hydrophobic α-helical domain located above the carboxyl-terminal ends of the strands of the barrel. Although the catalytic core is similar to that of the PutA PRODH domain, the FAD conformation of T. thermophilus PRODH is remarkably different and likely reflects unique requirements for membrane association and communication with P5CDH. Also, the FAD of T. thermophilus PRODH is highly solvent-exposed compared with PutA due to a 4-Å shift of helix 8. Structure-based sequence analysis of the PutA/PRODH family led us to identify nine conserved motifs involved in cofactor and substrate recognition. Biochemical studies show that the midpoint potential of the FAD is -75 mV and the kinetic parameters for proline are Km = 27 mm and kcat = 13 s-1. 3,4-Dehydro-l-proline was found to be an efficient substrate, and l-tetrahydro-2-furoic acid is a competitive inhibitor (KI = 1.0 mm). Finally, we demonstrate that T. thermophilus PRODH reacts with O2 producing superoxide. This is significant because superoxide production underlies the role of human PRODH in p53-mediated apoptosis, implying commonalities between eukaryotic and bacterial monofunctional PRODHs.

Tomato QM-Like Protein Protects Saccharomyces cerevisiae Cells against Oxidative Stress by Regulating Intracellular Proline Levels

ABSTRACT Exogenous proline can protect cells of Saccharomyces cerevisiae from oxidative stress. We altered intracellular proline levels by overexpressing the proline dehydrogenase gene (PUT1) of S. cerevisiae. Put1p performs the first enzymatic step of proline degradation in S. cerevisiae. Overexpression of Put1p results in low proline levels and hypersensitivity to oxidants, such as hydrogen peroxide and paraquat. A put1-disrupted yeast mutant deficient in Put1p activity has increased protection from oxidative stress and increased proline levels. Following a conditional life/death screen in yeast, we identified a tomato (Lycopersicon esculentum) gene encoding a QM-like protein (tQM) and found that stable expression of tQM in the Put1p-overexpressing strain conferred protection against oxidative damage from H2O2, paraquat, and heat. This protection was correlated with reactive oxygen species (ROS) reduction and increased proline accumulation. A yeast two-hybrid system assay was used to show that tQM physically interacts with Put1p in yeast, suggesting that tQM is directly involved in modulating proline levels. tQM also can rescue yeast from the lethality mediated by the mammalian proapoptotic protein Bax, through the inhibition of ROS generation. Our results suggest that tQM is a component of various stress response pathways and may function in proline-mediated stress tolerance in plants.

Crystal structure of the bifunctional proline utilization A flavoenzyme from Bradyrhizobium japonicum

The bifunctional proline catabolic flavoenzyme, proline utilization A (PutA), catalyzes the oxidation of proline to glutamate via the sequential activities of FAD-dependent proline dehydrogenase (PRODH) and NAD+-dependent Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Although structures for some of the domains of PutA are known, a structure for the full-length protein has not previously been solved. Here we report the 2.1 Å resolution crystal structure of PutA from Bradyrhizobium japonicum, along with data from small-angle x-ray scattering, analytical ultracentrifugation, and steady-state and rapid-reaction kinetics. PutA forms a ring-shaped tetramer in solution having a diameter of 150 Å. Within each protomer, the PRODH and P5CDH active sites face each other at a distance of 41 Å and are connected by a large, irregularly shaped cavity. Kinetics measurements show that glutamate production occurs without a lag phase, suggesting that the intermediate, Δ1-pyrroline-5-carboxylate, is preferably transferred to the P5CDH domain rather than released into the bulk medium. The structural and kinetic data imply that the cavity serves both as a microscopic vessel for the hydrolysis of Δ1-pyrroline-5-carboxylate to glutamate semialdehyde and a protected conduit for the transport of glutamate semialdehyde to the P5CDH active site.

Proline dehydrogenase is essential for proline protection against hydrogen peroxide induced cell death

Proline metabolism has an underlying role in apoptotic signaling that influences tumorigenesis. Proline is oxidized to glutamate in the mitochondria, with the rate-limiting step catalyzed by proline dehydrogenase (PRODH). PRODH expression is inducible by p53, leading to increased proline oxidation, reactive oxygen species formation, and induction of apoptosis. Paradoxical to its role in apoptosis, proline also protects cells against oxidative stress. Here we explore the mechanism of proline protection against hydrogen peroxide stress in melanoma WM35 cells. Treatment of WM35 cells with proline significantly increased cell viability, diminished oxidative damage of cellular lipids and proteins, and maintained ATP and NADPH levels after exposure to hydrogen peroxide. Inhibition or siRNA-mediated knockdown of PRODH abolished proline protection against oxidative stress, whereas knockdown of Δ(1)-pyrroline-5-carboxylate reductase, a key enzyme in proline biosynthesis, had no impact on proline protection. Potential linkages between proline metabolism and signaling pathways were explored. The combined inhibition of the mammalian target of rapamycin complex 1 (mTORC1) and mTORC2 eliminated proline protection. A significant increase in Akt activation was observed in proline-treated cells after hydrogen peroxide stress along with a corresponding increase in the phosphorylation of the forkhead transcription factor class O3a (FoxO3a). The role of PRODH in proline-mediated protection was validated in the prostate carcinoma cell line PC3. Knockdown of PRODH in PC3 cells attenuated phosphorylated levels of Akt and FoxO3a and decreased cell survival during hydrogen peroxide stress. The results provide evidence that PRODH is essential in proline protection against hydrogen peroxide-mediated cell death and that proline/PRODH helps activate Akt in cancer cells.

Modulation of the heme electronic structure and cystathionine β-synthase activity by second coordination sphere ligands: The role of heme ligand switching in redox regulation

In humans, cystathionine beta-synthase (CBS) is a hemeprotein, which catalyzes a pyridoxal phosphate (PLP)-dependent condensation reaction. Changes in the heme environment are communicated to the active site, which is approximately 20A away. In this study, we have examined the role of H67 and R266, which are in the second coordination sphere of the heme ligands, H65 and C52, respectively, in modulating the hemes electronic properties and in transmitting information between the heme and active sites. While the H67A mutation is comparable to wild-type CBS, interesting differences are revealed by mutations at the R266 site. The pathogenic mutant, R266K, is moderately PLP-responsive while the R266M mutation shows dramatic differences in the ferrous state. The electrostatic interaction between C52 and R266 is critical for stabilizing the ferrous heme and its disruption leads to the facile formation of a 424nm (C-424) absorbing ferrous species, which is inactive, compared to the active 449nm ferrous species for wild-type CBS. Resonance Raman studies on the R266M mutant reveal that the kinetics of C52 rebinding after Fe-CO photolysis are comparable to that of wild-type CBS. EXAFS studies on C-424 CBS are consistent with the presence of two axial N/O low Z scatters with only one being a rigid unit of a histidine residue while the other could be a solvent molecule, an oxygen atom from the peptide backbone or a side chain nitrogen. The redox potential for the heme in full-length CBS is -350+/-4mV and is substantially lower than the value of -287+/-2mV determined for truncated CBS. A redox-regulated ligand change has the potential to serve as an allosteric on/off switch in human CBS and the second sphere ligand, R266, plays an important role in this transition.

Oxygen reactivity of PutA from Helicobacter species and proline-linked oxidative stress

Proline is converted to glutamate in two successive steps by the proline utilization A (PutA) flavoenzyme in gram-negative bacteria. PutA contains a proline dehydrogenase domain that catalyzes the flavin adenine dinucleotide (FAD)-dependent oxidation of proline to delta1-pyrroline-5-carboxylate (P5C) and a P5C dehydrogenase domain that catalyzes the NAD+-dependent oxidation of P5C to glutamate. Here, we characterize PutA from Helicobacter hepaticus (PutA(Hh)) and Helicobacter pylori (PutA(Hp)) to provide new insights into proline metabolism in these gastrointestinal pathogens. Both PutA(Hh) and PutA(Hp) lack DNA binding activity, in contrast to PutA from Escherichia coli (PutA(Ec)), which both regulates and catalyzes proline utilization. PutA(Hh) and PutA(Hp) display catalytic activities similar to that of PutA(Ec) but have higher oxygen reactivity. PutA(Hh) and PutA(Hp) exhibit 100-fold-higher turnover numbers (approximately 30 min(-1)) than PutA(Ec) (<0. 3 min(-1)) using oxygen as an electron acceptor during catalytic turnover with proline. Consistent with increased oxygen reactivity, PutA(Hh) forms a reversible FAD-sulfite adduct. The significance of increased oxygen reactivity in PutA(Hh) and PutA(Hp) was probed by oxidative stress studies in E. coli. Expression of PutA(Ec) and PutA from Bradyrhizobium japonicum, which exhibit low oxygen reactivity, does not diminish stress survival rates of E. coli cell cultures. In contrast, PutA(Hp) and PutA(Hh) expression dramatically reduces E. coli cell survival and is correlated with relatively lower proline levels and increased hydrogen peroxide formation. The discovery of reduced oxygen species formation by PutA suggests that proline catabolism may influence redox homeostasis in the ecological niches of these Helicobacter species.

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

Significance Bifunctional enzymes catalyze two sequential reactions of a metabolic pathway using spatially separated and covalently connected active sites. Combining two enzymes into one protein chain creates efficiency because the product of the first reaction can be shuttled directly to the active site, catalyzing the second reaction in a process called substrate channeling. Here, we use X-ray crystallography to elucidate the three-dimensional structural basis of substrate channeling in the proline catabolic pathway. Structures of the bifunctional enzyme proline utilization A (PutA) reveal an elaborate and dynamic tunnel system featuring a 75-Å-long substrate-channeling path that links the two active sites. Furthermore, we have identified the elusive quinone-binding site of PutA, which provides insight into how PutAs interact with the membrane. 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.

Characterization of a Helicobacter hepaticus putA mutant strain in host colonization and oxidative stress.

ABSTRACT Helicobacter hepaticus is a gram-negative, spiral-shaped microaerophilic bacterium associated with chronic intestinal infection leading to hepatitis and colonic and hepatic carcinomas in susceptible strains of mice. In the closely related human pathogen Helicobacter pylori, l-proline is a preferred respiratory substrate and is found at significantly high levels in the gastric juice of infected patients. A previous study of the proline catabolic PutA flavoenzymes from H. pylori and H. hepaticus revealed that Helicobacter PutA generates reactive oxygen species during proline oxidation by transferring electrons from reduced flavin to molecular oxygen. We further explored the preference for proline as a respiratory substrate and the potential impact of proline metabolism on the redox environment in Helicobacter species during host infection by disrupting the putA gene in H. hepaticus. The resulting putA knockout mutant strain was characterized by oxidative stress analysis and mouse infection studies. The putA mutant strain of H. hepaticus exhibited increased proline levels and resistance to oxidative stress relative to that of the wild-type strain, consistent with prolines role as an antioxidant. The significant increase in stress resistance was attributed to higher proline content, as no upregulation of antioxidant genes was observed for the putA mutant strain. The wild-type and putA mutant H. hepaticus strains displayed similar levels of infection in mice, but in mice challenged with the putA mutant strain, significantly reduced inflammation was observed, suggesting a role for proline metabolism in H. hepaticus pathogenicity in vivo.

Substrate channeling in proline metabolism.

Proline metabolism is an important pathway that has relevance in several cellular functions such as redox balance, apoptosis, and cell survival. Results from different groups have indicated that substrate channeling of proline metabolic intermediates may be a critical mechanism. One intermediate is pyrroline-5-carboxylate (P5C), which upon hydrolysis opens to glutamic semialdehyde (GSA). Recent structural and kinetic evidence indicate substrate channeling of P5C/GSA occurs in the proline catabolic pathway between the proline dehydrogenase and P5C dehydrogenase active sites of bifunctional proline utilization A (PutA). Substrate channeling in PutA is proposed to facilitate the hydrolysis of P5C to GSA which is unfavorable at physiological pH. The second intermediate, gamma-glutamyl phosphate, is part of the proline biosynthetic pathway and is extremely labile. Substrate channeling of gamma-glutamyl phosphate is thought to be necessary to protect it from bulk solvent. Because of the unfavorable equilibrium of P5C/GSA and the reactivity of gamma-glutamyl phosphate, substrate channeling likely improves the efficiency of proline metabolism. Here, we outline general strategies for testing substrate channeling and review the evidence for channeling in proline metabolism.