Rid Protein Research Articles

DadY (PA5303) is required for fitness of Pseudomonas aeruginosa when growth is dependent on alanine catabolism.

Pseudomonas aeruginosa inhabits diverse environmental niches that can have varying nutrient composition. The ubiquity of this organism is facilitated by a metabolic strategy that preferentially utilizes low-energy, non-fermentable organic acids, such as amino acids, rather than the high-energy sugars preferred by many other microbes. The amino acid alanine is among the preferred substrates of P. aeruginosa. The dad locus encodes the constituents of the alanine catabolic pathway of P. aeruginosa. Physiological roles for DadR (AsnC-type transcriptional activator), DadX (alanine racemase), and DadA (D-amino acid dehydrogenase) have been defined in this pathway. An additional protein, PA5303, is encoded in the dad locus in P. aeruginosa. PA5303 is a member of the ubiquitous Rid protein superfamily and is designated DadY based on the data presented herein. Despite its conservation in numerous Pseudomonas species and membership in the Rid superfamily, no physiological function has been assigned to DadY. In the present study, we demonstrate that DadA releases imino-alanine that can be deaminated by DadY in vitro. While DadY was not required for alanine catabolism in monoculture, dadY mutants had a dramatic fitness defect in competition with wild-type P. aeruginosa when alanine served as the sole carbon or nitrogen source. The data presented herein support a model in which DadY facilitates flux through the alanine catabolic pathway by removing the imine intermediate generated by DadA. Functional characterization of DadY contributes to our understanding of the role of the broadly conserved Rid family members.

The Rid family member RutC of Escherichia coli is a 3-aminoacrylate deaminase

The Rid protein family (PF14588, IPR006175) is divided into nine subfamilies, of which only the RidA subfamily has been characterized biochemically. RutC, the founding member of one subfamily, is encoded in the pyrimidine utilization (rut) operon that encodes a pathway that allows Escherichia coli to use uracil as a sole nitrogen source. Results reported herein demonstrate that RutC has 3-aminoacrylate deaminase activity and facilitates one of the reactions previously presumed to occur spontaneously in vivo. RutC was active with several enamine–imine substrates, showing similarities and differences in substrate specificity with the canonical member of the Rid superfamily, Salmonella enterica RidA. Under standard laboratory conditions, a Rut pathway lacking RutC generates sufficient nitrogen from uracil for growth of E. coli. These results support a revised model of the Rut pathway and provide evidence that Rid proteins may modulate metabolic fitness, rather than catalyzing essential functions.

Putative Horizontally Acquired Genes, Highly Transcribed during Yersinia pestis Flea Infection, Are Induced by Hyperosmotic Stress and Function in Aromatic Amino Acid Metabolism.

While alternating between insects and mammals during its life cycle, Yersinia pestis, the flea-transmitted bacterium that causes plague, regulates its gene expression appropriately to adapt to these two physiologically disparate host environments. In fleas competent to transmit Y. pestis, low-GC-content genes y3555, y3551, and y3550 are highly transcribed, suggesting that these genes have a highly prioritized role in flea infection. Here, we demonstrate that y3555, y3551, and y3550 are transcribed as part of a single polycistronic mRNA comprising the y3555, y3554, y3553, y355x, y3551, and y3550 genes. Additionally, y355x-y3551-y3550 compose another operon, while y3550 can be also transcribed as a monocistronic mRNA. The expression of these genes is induced by hyperosmotic salinity stress, which serves as an explicit environmental stimulus that initiates transcriptional activity from the predicted y3550 promoter. Y3555 has homology to pyridoxal 5'-phosphate (PLP)-dependent aromatic aminotransferases, while Y3550 and Y3551 are homologous to the Rid protein superfamily (YjgF/YER057c/UK114) members that forestall damage caused by reactive intermediates formed during PLP-dependent enzymatic activity. We demonstrate that y3551 specifically encodes an archetypal RidA protein with 2-aminoacrylate deaminase activity but Y3550 lacks Rid deaminase function. Heterologous expression of y3555 generates a critical aspartate requirement in a Salmonella entericaaspC mutant, while its in vitro expression, and specifically its heterologous coexpression with y3550, enhances the growth rate of an Escherichia coli ΔaspC ΔtyrB mutant in a defined minimal amino acid-supplemented medium. Our data suggest that the y3555, y3551, and y3550 genes operate cooperatively to optimize aromatic amino acid metabolism and are induced under conditions of hyperosmotic salinity stress.IMPORTANCE Distinct gene repertoires are expressed during Y. pestis infection of its flea and mammalian hosts. The functions of many of these genes remain predicted or unknown, necessitating their characterization, as this may provide a better understanding of Y. pestis specialized biological adaptations to the discrete environments of its two hosts. This study provides functional context to adjacently clustered horizontally acquired genes predominantly expressed in the flea host by deciphering their fundamental processes with regard to (i) transcriptional organization, (ii) transcription activation signals, and (iii) biochemical function. Our data support a role for these genes in osmoadaptation and aromatic amino acid metabolism, highlighting these as preferential processes by which Y. pestis gene expression is modulated during flea infection.

Imine Deaminase Activity and Conformational Stability of UK114, the Mammalian Member of the Rid Protein Family Active in Amino Acid Metabolism.

Reactive intermediate deaminase (Rid) protein family is a recently discovered group of enzymes that is conserved in all domains of life and is proposed to play a role in the detoxification of reactive enamines/imines. UK114, the mammalian member of RidA subfamily, was identified in the early 90s as a component of perchloric acid-soluble extracts from goat liver and exhibited immunomodulatory properties. Multiple activities were attributed to this protein, but its function is still unclear. This work addressed the question of whether UK114 is a Rid enzyme. Biochemical analyses demonstrated that UK114 hydrolyzes α-imino acids generated by l- or d-amino acid oxidases with a preference for those deriving from Ala > Leu = l-Met > l-Gln, whereas it was poorly active on l-Phe and l-His. Circular Dichroism (CD) analyses of UK114 conformational stability highlighted its remarkable resistance to thermal unfolding, even at high urea concentrations. The half-life of heat inactivation at 95 °C, measured from CD and activity data, was about 3.5 h. The unusual conformational stability of UK114 could be relevant in the frame of a future evaluation of its immunogenic properties. In conclusion, mammalian UK114 proteins are RidA enzymes that may play an important role in metabolism homeostasis also in these organisms.

Members of the Rid protein family have broad imine deaminase activity and can accelerate the Pseudomonas aeruginosa D-arginine dehydrogenase (DauA) reaction in vitro.

The Rid (YjgF/YER057c/UK114) protein family is a group of small, sequence diverse proteins that consists of eight subfamilies. The archetypal RidA subfamily is found in all domains, while the Rid1-7 subfamilies are present only in prokaryotes. Bacterial genomes often encode multiple members of the Rid superfamily. The best characterized member of this protein family, RidA from Salmonella enterica, is a deaminase that quenches the reactive metabolite 2-aminoacrylate generated by pyridoxal 5’-phosphate-dependent enzymes and ultimately spares certain enzymes from damage. The accumulation of 2-aminoacrylate can damage enzymes and lead to growth defects in bacteria, plants, and yeast. While all subfamily members have been annotated as imine deaminases based on the RidA characterization, experimental evidence to support this annotation exists for a single protein outside the RidA subfamily. Here we report that six proteins, spanning Rid subfamilies 1–3, deaminate a variety of imine/enamine substrates with differing specific activities. Proteins from the Rid2 and Rid3 subfamilies, but not from the RidA and Rid1 subfamilies deaminated iminoarginine, generated in situ by the Pseudomonas aeruginosa D-arginine dehydrogenase DauA. These data biochemically distinguished the subfamilies and showed Rid proteins have activity on a metabolite that is physiologically relevant in Pseudomonas and other bacteria.

2-Aminoacrylate Stress Induces a Context-Dependent Glycine Requirement in ridA Strains of Salmonella enterica.

The reactive enamine 2-aminoacrylate (2AA) is a metabolic stressor capable of damaging cellular components. Members of the broadly conserved Rid (RidA/YER057c/UK114) protein family mitigate 2AA stress in vivo by facilitating enamine and/or imine hydrolysis. Previous work showed that 2AA accumulation in ridA strains of Salmonella enterica led to the inactivation of multiple target enzymes, including serine hydroxymethyltransferase (GlyA). However, the specific cause of a ridA strain's inability to grow during periods of 2AA stress had yet to be determined. Work presented here shows that glycine supplementation suppressed all 2AA-dependent ridA strain growth defects described to date. Depending on the metabolic context, glycine appeared to suppress ridA strain growth defects by eliciting a GcvB small RNA-dependent regulatory response or by serving as a precursor to one-carbon units produced by the glycine cleavage complex (GCV). In either case, the data suggest that GlyA is the most physiologically sensitive target of 2AA inactivation in S. enterica. The universally conserved nature of GlyA among free-living organisms highlights the importance of RidA in mitigating 2AA stress. The RidA stress response prevents 2-aminoacrylate (2AA) damage from occurring in prokaryotes and eukaryotes alike. 2AA inactivation of serine hydroxymethyltransferase (GlyA) from Salmonella enterica restricts glycine and one-carbon production, ultimately reducing fitness of the organism. The cooccurrence of genes encoding 2AA production enzymes and serine hydroxy-methyltransferase (SHMT) in many genomes may in part underlie the evolutionary selection for Rid proteins to maintain appropriate glycine and one-carbon metabolism throughout life.

From microbiology to cancer biology: the Rid protein family prevents cellular damage caused by endogenously generated reactive nitrogen species.

The Rid family of proteins is highly conserved and broadly distributed throughout the domains of life. Genetic and biochemical studies, primarily in Salmonella enterica, have defined a role for RidA in responding to endogenously generated reactive metabolites. The data show that 2-aminoacrylate (2AA), a reactive enamine intermediate generated by some pyridoxal 5'-phosphate-dependent enzymes, accumulates in the absence of RidA. The accumulation of 2AA leads to covalent modification and inactivation of several enzymes involved in essential metabolic processes. This review describes the 2AA hydrolyzing activity of RidA and the effect of this biochemical activity on the metabolic network, which impacts organism fitness. The reported activity of RidA and the consequences encountered in vivo when RidA is absent have challenged fundamental assumptions in enzymology, biochemistry and cell metabolism regarding the fate of transiently generated reactive enamine intermediates. The current understanding of RidA in Salmonella and the broad distribution of Rid family proteins provide exciting opportunities for future studies to define metabolic roles of Rid family members from microbes to man.