Staphylococcus aureus does not synthesize arginine from proline under physiological conditions

The Gram-positive pathogen Staphylococcus aureus is the only bacterium known to synthesize arginine from proline via the arginine-proline interconversion pathway, despite having genes for the well-conserved glutamate pathway. Since the proline-arginine interconversion pathway is repressed by CcpA-mediated carbon catabolite repression (CCR), CCR has been attributed to the arginine auxotrophy of S. aureus. Using ribose as a secondary carbon source, here, we demonstrate that S. aureus arginine auxotrophy is not due to CCR but due to the inadequate concentration of proline degradation product. Proline is degraded by proline dehydrogenase (PutA) into pyrroline-5-carboxylate (P5C). Although the PutA expression was fully induced by ribose, the P5C concentration remained insufficient to support arginine synthesis because P5C was constantly consumed by the P5C reductase ProC. When the P5C concentration was artificially increased by either PutA overexpression or proC-deletion, S. aureus could synthesize arginine from proline regardless of carbon source. In contrast, when the P5C concentration was reduced by overexpression of proC, it inhibited the growth of the ccpA-deletion mutant without arginine. Intriguingly, the ectopic expression of the glutamate pathway enzymes converted S. aureus into arginine prototroph. In an animal experiment, the arginine-proline interconversion pathway was not required for the survival of S. aureus. Based on these results, we concluded that S. aureus does not synthesize arginine from proline under physiological conditions. We also propose that arginine auxotrophy of S. aureus is not due to the CcpA-mediated CCR but due to the inactivity of the conserved glutamate pathway.

Structure-Affinity Relationships of Reversible Proline Analog Inhibitors Targeting Proline Dehydrogenase

Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the FAD-dependent oxidation of L-proline to Δ1-pyrroline-5-carboxylate. PRODH plays a central role in the metabolic rewiring of cancer cells, which...

Higher expression of proline dehydrogenase altered mitochondrial function and increased Trypanosoma cruzi differentiation in vitro and in the insect vector

The pathogenic protist Trypanosoma cruzi uses kissing bugs as intermediate hosts that vectorize the infection among mammals. This parasite oxidizes proline to glutamate through two enzymatic steps and one nonenzymatic step. In insect vectors, T. cruzi differentiates from a noninfective replicating form to nonproliferative infective forms. Proline sustains this differentiation, but to date, a link between proline metabolism and differentiation has not been established. In T. cruzi, the enzymatic steps of the proline-glutamate oxidation pathway are catalysed exclusively by the mitochondrial enzymes proline dehydrogenase [TcPRODH, EC: 1.5.5.2] and D1-pyrroline-5-carboxylate dehydrogenase [TcP5CDH, EC: 1.2.1.88]. Both enzymatic steps produce reducing equivalents that are able to directly feed the mitochondrial electron transport chain (ETC) and thus produce ATP. In this study, we demonstrate the contribution of each enzyme of the proline-glutamate pathway to ATP production. In addition, we show that parasites overexpressing these enzymes produce increased levels of H2O2, but only those overexpressing TcP5CDH produce increased levels of superoxide anion. We show that parasites overexpressing TcPRODH, but not parasites overexpressing TcP5CDH, exhibit a higher rate of differentiation into metacyclic trypomastigotes in vitro. Finally, insect hosts infected with parasites overexpressing TcPRODH showed a diminished parasitic load but a higher percent of metacyclic trypomastigotes, when compared with controls. Our data show that parasites overexpressing both, PRODH and P5CDH had increased mitochondrial functions that orchestrated different oxygen signalling, resulting in different outcomes in relation to the efficiency of parasitic differentiation in the invertebrate host.

. Genes of proline metabolism in biotechnology of increasing wheat osmostability

Aim. To analyse effectiveness of introduced genes of ornithine-δ-aminotransferase (oat) Medicago truncatula and fragments of two copies of first exon and intron of the gene prolinedehydrogenase (pdh) of Arabidopsis thaliana, that form double stranded RNA suppressor of gene of the prolinedehydrogenase wheat, in the increase of her productivity for the actions ofosmotic stress. Methods. Determination of the content of free L-proline (Pro); of the activity of the enzyme ornithine-δ-aminotransferase (OAT), the activity of proline dehydrogenase (PDG), indicators of crop structure. Results. It was found that wheat plants with an additional copy of the oat gene are characterized by increased OAT activity, which is not reflected in the Pro content. Analysis of plants with integrated elements that form a double-stranded RNA suppressor of the pdh gene found a decrease in PDG activity and an increase in Pro content. It was found that T2 generation of biotechnological plants UK 322/17 with suppressor of the pdh gene and UK 95/17 with an additional copy of the oat gene, in drought conditions were characterized by higher rates for a number of crop elements compared to their original forms. Conclusions. Increased expression of the oat gene leads to improved wheat growth, which has a positive effect on plant productivity in conditions of water deficiency. Partial suppression of the proline dehydrogenase gene causes improved performance due to the increased content of free proline during drought. Keywords: wheat, transgenic plants, proline, ornithine-δ-aminotransferase, proline dehydrogenase, soil drought, grain productivity.

N-Propargylglycine: a unique suicide inhibitor of proline dehydrogenase with anticancer activity and brain-enhancing mitohormesis properties

Proline dehydrogenase (PRODH) is a mitochondrial inner membrane flavoprotein critical for cancer cell survival under stress conditions and newly recognized as a potential target for cancer drug development. Reversible (competitive) and irreversible (suicide) inhibitors of PRODH have been shown in vivo to inhibit cancer cell growth with excellent host tolerance. Surprisingly, the PRODH suicide inhibitor N-propargylglycine (N-PPG) also induces rapid decay of PRODH with concordant upregulation of mitochondrial chaperones (HSP-60, GRP-75) and the inner membrane protease YME1L1, signifying activation of the mitochondrial unfolded protein response (UPRmt) independent of anticancer activity. The present study was undertaken to address two aims: (i) use PRODH overexpressing human cancer cells (ZR-75-1) to confirm the UPRmt inducing properties of N-PPG relative to another equipotent irreversible PRODH inhibitor, thiazolidine-2-carboxylate (T2C); and (ii) employ biochemical and transcriptomic approaches to determine if orally administered N-PPG can penetrate the blood–brain barrier, essential for its future use as a brain cancer therapeutic, and also potentially protect normal brain tissue by inducing mitohormesis. Oral daily treatments of N-PPG produced a dose-dependent decline in brain mitochondrial PRODH protein without detectable impairment in mouse health; furthermore, mice repeatedly dosed with 50 mg/kg N-PPG showed increased brain expression of the mitohormesis associated protease, YME1L1. Whole brain transcriptome (RNAseq) analyses of these mice revealed significant gene set enrichment in N-PPG stimulated neural processes (FDR p < 0.05). Given this in vivo evidence of brain bioavailability and neural mitohormesis induction, N-PPG appears to be unique among anticancer agents and should be evaluated for repurposing as a pharmaceutical capable of mitigating the proteotoxic mechanisms driving neurodegenerative disorders.

In Silico Structural Modeling of Wildtype and Mutant PRODH Proteins Involved in Psychiatric Disorder.

Proline dehydrogenase is an important mitochondrial enzyme that is encoded by the PRODH gene. Biologically, a mutation in this gene affects the activity of proline dehydrogenase enzyme that is normally involved in conversion of proline to glutamate. However, its reduced or null activity leads to excess quantity of proline in the body, which results in different psychiatric phenotypes along with intellectual disability. In the present study, we performed in silico analysis on all reported mutations of PRODH. The 3D models of normal and mutant PRODH were predicted using I-TASSER. The predicted structures were visualized and superimposed using chimera 1.13.1. The CASTp was used to identify active sites in modelled proteins. Protein-protein docking was done with Cluspro, while protein-substrate docking was done with Auto Dock 1.5.6 and-MGL tools and the results were visualized using LigPlus+ v.2.2 and Discovery studio 2020 respectively. Alignment of 3D models (mutant with wildtype) revealed that Arg185Gln (73.83 % ) and Gln19Ter (6.25% ) had the highest and lowest similarity indices, respectively. Enzyme pocket prediction identified three largest sites, with the second largest active site pocket containing substrate proline binding residues Leu527, Tyr548, and Arg563. Moreover, docking of mutant and wildtype PRODH with its close interactor “ALDH4A1” showed differences with respect to number, position, and nature of interacting amino acids residues. We observed that the nature of amino acid substitution and the number of bonds affect the binding of proline molecule with proline dehydrogenase enzyme, and therefore, affect its biological activity.

Collagen metabolism as a regulator of proline dehydrogenase/proline oxidase-dependent apoptosis/autophagy

Recent studies on the regulatory role of amino acids in cell metabolism have focused on the functional significance of proline degradation. The process is catalysed by proline dehydrogenase/proline oxidase (PRODH/POX), a mitochondrial flavin-dependent enzyme converting proline into ∆1-pyrroline-5-carboxylate (P5C). During this process, electrons are transferred to electron transport chain producing ATP for survival or they directly reduce oxygen, producing reactive oxygen species (ROS) inducing apoptosis/autophagy. However, the mechanism for switching survival/apoptosis mode is unknown. Although PRODH/POX activity and energetic metabolism were suggested as an underlying mechanism for the survival/apoptosis switch, proline availability for this enzyme is also important. Proline availability is regulated by prolidase (proline supporting enzyme), collagen biosynthesis (proline utilizing process) and proline synthesis from glutamine, glutamate, α-ketoglutarate (α-KG) and ornithine. Proline availability is dependent on the rate of glycolysis, TCA and urea cycles, proline metabolism, collagen biosynthesis and its degradation. It is well established that proline synthesis enzymes, P5C synthetase and P5C reductase as well as collagen prolyl hydroxylases are up-regulated in most of cancer types and control rates of collagen biosynthesis. Up-regulation of collagen prolyl hydroxylase and its exhaustion of ascorbate and α-KG may compete with DNA and histone demethylases (that require the same cofactors) to influence metabolic epigenetics. This knowledge led us to hypothesize that up-regulation of prolidase and PRODH/POX with inhibition of collagen biosynthesis may represent potential pharmacotherapeutic approach to induce apoptosis or autophagic death in cancer cells. These aspects of proline metabolism are discussed in the review as an approach to understand complex regulatory mechanisms driving PRODH/POX-dependent apoptosis/survival.