Dustin Ernst

Crystal Structure of the First Eubacterial Mre11 Nuclease Reveals Novel Features that May Discriminate Substrates During DNA Repair

Mre11 nuclease plays a central role in the repair of cytotoxic and mutagenic DNA double-strand breaks. As X-ray structural information has been available only for the Pyrococcus furiosus enzyme (PfMre11), the conserved and variable features of this nuclease across the domains of life have not been experimentally defined. Our crystal structure and biochemical studies demonstrate that TM1635 from Thermotoga maritima, originally annotated as a putative nuclease, is an Mre11 endo/exonuclease (TmMre11) and the first such structure from eubacteria. TmMre11 and PfMre11 display similar overall structures, despite sequence identity in the twilight zone of only approximately 20%. However, they differ substantially in their DNA-specificity domains and in their dimeric organization. Residues in the nuclease domain are highly conserved, but those in the DNA-specificity domain are not. The structural differences likely affect how Mre11 from different organisms recognize and interact with single-stranded DNA, double-stranded DNA and DNA hairpin structures during DNA repair. The TmMre11 nuclease active site has no bound metal ions, but is conserved in sequence and structure with the exception of a histidine that is important in PfMre11 nuclease activity. Nevertheless, biochemical characterization confirms that TmMre11 possesses both endonuclease and exonuclease activities on single-stranded and double-stranded DNA substrates, respectively.

Bacterial Pleckstrin Homology Domains: a Prokaryotic Origin for the Ph Domain

Pleckstrin homology (PH) domains have been identified only in eukaryotic proteins to date. We have determined crystal structures for three members of an uncharacterized protein family (Pfam PF08000), which provide compelling evidence for the existence of PH-like domains in bacteria (PHb). The first two structures contain a single PHb domain that forms a dome-shaped, oligomeric ring with C5 symmetry. The third structure has an additional helical hairpin attached at the C-terminus and forms a similar but much larger ring with C12 symmetry. Thus, both molecular assemblies exhibit rare, higher-order, cyclic symmetry but preserve a similar arrangement of their PHb domains, which gives rise to a conserved hydrophilic surface at the intersection of the β-strands of adjacent protomers that likely mediates protein–protein interactions. As a result of these structures, additional families of PHb domains were identified, suggesting that PH domains are much more widespread than originally anticipated. Thus, rather than being a eukaryotic innovation, the PH domain superfamily appears to have existed before prokaryotes and eukaryotes diverged.

Structural and Functional Characterizations of SsgB, a Conserved Activator of Developmental Cell Division in Morphologically Complex Actinomycetes

SsgA-like proteins (SALPs) are a family of homologous cell division-related proteins that occur exclusively in morphologically complex actinomycetes. We show that SsgB, a subfamily of SALPs, is the archetypal SALP that is functionally conserved in all sporulating actinomycetes. Sporulation-specific cell division of Streptomyces coelicolor ssgB mutants is restored by introduction of distant ssgB orthologues from other actinomycetes. Interestingly, the number of septa (and spores) of the complemented null mutants is dictated by the specific ssgB orthologue that is expressed. The crystal structure of the SsgB from Thermobifida fusca was determined at 2.6 Å resolution and represents the first structure for this family. The structure revealed similarities to a class of eukaryotic “whirly” single-stranded DNA/RNA-binding proteins. However, the electro-negative surface of the SALPs suggests that neither SsgB nor any of the other SALPs are likely to interact with nucleotide substrates. Instead, we show that a conserved hydrophobic surface is likely to be important for SALP function and suggest that proteins are the likely binding partners.

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

UNLABELLED 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 strains 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. IMPORTANCE 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.

Crystal structure of a novel Sm-like protein of putative cyanophage origin at 2.60 Å resolution

ECX21941 represents a very large family (over 600 members) of novel, ocean metagenome‐specific proteins identified by clustering of the dataset from the Global Ocean Sampling expedition. The crystal structure of ECX21941 reveals unexpected similarity to Sm/LSm proteins, which are important RNA‐binding proteins, despite no detectable sequence similarity. The ECX21941 protein assembles as a homopentamer in solution and in the crystal structure when expressed in Escherichia coli and represents the first pentameric structure for this Sm/LSm family of proteins, although the actual oligomeric form in vivo is currently not known. The genomic neighborhood analysis of ECX21941 and its homologs combined with sequence similarity searches suggest a cyanophage origin for this protein. The specific functions of members of this family are unknown, but our structure analysis of ECX21941 indicates nucleic acid‐binding capabilities and suggests a role in RNA and/or DNA processing. Proteins 2009.

L‐2,3‐diaminopropionate generates diverse metabolic stresses in Salmonella enterica

Unchecked amino acid accumulation in living cells has the potential to cause stress by disrupting normal metabolic processes. Thus, many organisms have evolved degradation strategies that prevent endogenous accumulation of amino acids. L‐2,3‐diaminopropionate (Dap) is a non‐protein amino acid produced in nature where it serves as a precursor to siderophores, neurotoxins and antibiotics. Dap accumulation in Salmonella enterica was previously shown to inhibit growth by unknown mechanisms. The production of diaminopropionate ammonia‐lyase (DpaL) alleviated Dap toxicity in S. enterica by catalyzing the degradation of Dap to pyruvate and ammonia. Here, we demonstrate that Dap accumulation in S. enterica elicits a proline requirement for growth and specifically inhibits coenzyme A and isoleucine biosynthesis. Additionally, we establish that the DpaL‐dependent degradation of Dap to pyruvate proceeds through an unbound 2‐aminoacrylate (2AA) intermediate, thus contributing to 2AA stress inside the cell. The reactive intermediate deaminase, RidA, is shown to prevent 2AA damage caused by DpaL‐dependent Dap degradation by enhancing the rate of 2AA hydrolysis. The results presented herein inform our understanding of the effects Dap has on metabolism in S. enterica, and likely other organisms, and highlight the critical role played by RidA in preventing 2AA stress stemming from Dap detoxification.

Structures of the first representatives of Pfam family PF06938 (DUF1285) reveal a new fold with repeated structural motifs and possible involvement in signal transduction.

The crystal structures of SPO0140 and Sbal_2486 revealed a two-domain structure that adopts a novel fold. Analysis of the interdomain cleft suggests a nucleotide-based ligand with a genome context indicating signaling as a possible role for this family.

The structure of BVU2987 from Bacteroides vulgatus reveals a superfamily of bacterial periplasmic proteins with possible inhibitory function

The crystal structure of the BVU2987 gene product from B. vulgatus (UniProt A6L4L1) reveals that members of the new Pfam family PF11396 (domain of unknown function; DUF2874) are similar to β-lactamase inhibitor protein and YpmB.

Mmf1p Couples Amino Acid Metabolism to Mitochondrial DNA Maintenance in Saccharomyces cerevisiae

ABSTRACT A variety of metabolic deficiencies and human diseases arise from the disruption of mitochondrial enzymes and/or loss of mitochondrial DNA. Mounting evidence shows that eukaryotes have conserved enzymes that prevent the accumulation of reactive metabolites that cause stress inside the mitochondrion. 2-Aminoacrylate is a reactive enamine generated by pyridoxal 5′-phosphate-dependent α,β-eliminases as an obligatory intermediate in the breakdown of serine. In prokaryotes, members of the broadly conserved RidA family (PF14588) prevent metabolic stress by deaminating 2-aminoacrylate to pyruvate. Here, we demonstrate that unmanaged 2-aminoacrylate accumulation in Saccharomyces cerevisiae mitochondria causes transient metabolic stress and the irreversible loss of mitochondrial DNA. The RidA family protein Mmf1p deaminates 2-aminoacrylate, preempting metabolic stress and loss of the mitochondrial genome. Disruption of the mitochondrial pyridoxal 5′-phosphate-dependent serine dehydratases (Ilv1p and Cha1p) prevents 2-aminoacrylate formation, avoiding stress in the absence of Mmf1p. Furthermore, chelation of iron in the growth medium improves maintenance of the mitochondrial genome in yeast challenged with 2-aminoacrylate, suggesting that 2-aminoacrylate-dependent loss of mitochondrial DNA is influenced by disruption of iron homeostasis. Taken together, the data indicate that Mmf1p indirectly contributes to mitochondrial DNA maintenance by preventing 2-aminoacrylate stress derived from mitochondrial amino acid metabolism. IMPORTANCE Deleterious reactive metabolites are produced as a consequence of many intracellular biochemical transformations. Importantly, reactive metabolites that appear short-lived in vitro have the potential to persist within intracellular environments, leading to pervasive cell damage and diminished fitness. To overcome metabolite damage, organisms utilize enzymatic reactive-metabolite defense systems to rid the cell of deleterious metabolites. In this report, we describe the importance of the RidA/YER057c/UK114 enamine/imine deaminase family in preventing 2-aminoacrylate stress in yeast. Saccharomyces cerevisiae lacking the enamine/imine deaminase Mmf1p was shown to experience pleiotropic growth defects and fails to maintain its mitochondrial genome. Our results provide the first line of evidence that uncontrolled 2-aminoacrylate stress derived from mitochondrial serine metabolism can negatively impact mitochondrial DNA maintenance in eukaryotes. Deleterious reactive metabolites are produced as a consequence of many intracellular biochemical transformations. Importantly, reactive metabolites that appear short-lived in vitro have the potential to persist within intracellular environments, leading to pervasive cell damage and diminished fitness. To overcome metabolite damage, organisms utilize enzymatic reactive-metabolite defense systems to rid the cell of deleterious metabolites. In this report, we describe the importance of the RidA/YER057c/UK114 enamine/imine deaminase family in preventing 2-aminoacrylate stress in yeast. Saccharomyces cerevisiae lacking the enamine/imine deaminase Mmf1p was shown to experience pleiotropic growth defects and fails to maintain its mitochondrial genome. Our results provide the first line of evidence that uncontrolled 2-aminoacrylate stress derived from mitochondrial serine metabolism can negatively impact mitochondrial DNA maintenance in eukaryotes.