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The AAA(+) family member KaiC is the central pacemaker for circadian rhythms in the cyanobacterium Synechococcus elongatus. Composed of two hexameric rings of adenosine triphosphatase (ATPase) domains with tightly coupled activities, KaiC undergoes a cycle of autophosphorylation and autodephosphorylation on its C-terminal (CII) domain that restricts binding of clock proteins on its N-terminal (CI) domain to the evening. Here, we use cryogenic-electron microscopy to investigate how daytime and nighttime states of CII regulate KaiB binding on CI. We find that the CII hexamer is destabilized during the day but takes on a rigidified C(2)-symmetric state at night, concomitant with ring-ring compression. Residues at the CI-CII interface are required for phospho-dependent KaiB association, coupling ATPase activity on CI to cooperative KaiB recruitment. Together, these studies clarify a key step in the regulation of cyanobacterial circadian rhythms by KaiC phosphorylation.

The RidA subfamily of the Rid (YjgF/YER057c/UK114) superfamily of proteins is broadly distributed and found in all domains of life. RidA proteins are enamine/imine deaminases. In the organisms that have been investigated, lack of RidA results in accumulation of the reactive enamine species 2-aminoacrylate (2AA) and/or its derivative imine 2-iminopropanoate (2IP). The accumulated enamine/imine species can damage specific pyridoxal phosphate (PLP)-dependent target enzymes. The metabolic imbalance resulting from the damaged enzymes is organism specific and based on metabolic network configuration. Saccharomyces cerevisiae encodes two RidA homologs, one localized to the cytosol and one to the mitochondria. The mitochondrial RidA homolog, Mmf1p, prevents enamine/imine stress and is important for normal growth and maintenance of mitochondrial DNA. Here, we show that Mmf1p is necessary for optimal heme biosynthesis. Biochemical and/or genetic data herein support a model in which accumulation of 2AA and or 2IP, in the absence of Mmf1p, inactivates Hem1p, a mitochondrially located PLP-dependent enzyme required for heme biosynthesis.

Circadian clocks control gene expression to provide an internal representation of local time. We report reconstitution of a complete cyanobacterial circadian clock in vitro, including the central oscillator, signal transduction pathways, downstream transcription factor, and promoter DNA. The entire system oscillates autonomously and remains phase coherent for many days with a fluorescence-based readout that enables real-time observation of each component simultaneously without user intervention. We identified the molecular basis for loss of cycling in an arrhythmic mutant and explored fundamental mechanisms of timekeeping in the cyanobacterial clock. We find that SasA, a circadian sensor histidine kinase associated with clock output, engages directly with KaiB on the KaiC hexamer to regulate period and amplitude of the central oscillator. SasA uses structural mimicry to cooperatively recruit the rare, fold-switched conformation of KaiB to the KaiC hexamer to form the nighttime repressive complex and enhance rhythmicity of the oscillator, particularly under limiting concentrations of KaiB. Thus, the expanded in vitro clock reveals previously unknown mechanisms by which the circadian system of cyanobacteria maintains the pace and rhythmicity under variable protein concentrations.

The AAA+ protein KaiC is the central pacemaker for cyanobacterial circadian rhythms. Composed of two hexameric rings with tightly coupled activities, KaiC undergoes changes in autophosphorylation on its C-terminal (CII) domain that restrict binding of of clock proteins on its N-terminal (CI) domain to the evening. Here, we use cryo-electron microscopy to investigate how daytime and nighttime states of CII regulate KaiB binding to CI. We find that the CII hexamer is destabilized during the day but takes on a rigidified C2-symmetric state at night,concomitant with ring-ring compression. Residues at the CI-CII interface are required for phospho-dependent KaiB association, coupling ATPase activity on CI to cooperative KaiB recruitment. Together these studies reveal how daily changes in KaiC phosphorylation regulate cyanobacterial circadian rhythms.One-Sentence SummaryCryo-EM structures of KaiC in its day and night states reveal the structural basis for assembly of clock regulatory complexes.

The histidine kinase SasA enhances robustness of circadian rhythms in the cyanobacterium S. elongatus by temporally controlling expression of the core clock components, kaiB and kaiC. Here we show that SasA also engages directly with KaiB and KaiC proteins to regulate the period and enhance robustness of the reconstituted circadian oscillator in vitro, particularly under limiting concentrations of KaiB. In contrast to its role regulating gene expression, oscillator function does not require SasA kinase activity; rather, SasA uses structural mimicry to cooperatively recruit the rare, fold-switched conformation of KaiB to the KaiC hexamer to form the nighttime repressive complex. Cooperativity gives way to competition with increasing concentrations of SasA to define a dynamic window by which SasA directly modulates clock robustness.One Sentence SummarySasA controls the assembly of clock protein complexes through a balance of cooperative and competitive interactions.

Circadian clocks control gene expression in the complex milieu of cells. Here, we reconstituted under defined conditions in vitro the cyanobacterial circadian clock system which includes an oscillator, signal-transduction pathways, transcription factor, and promoter DNA. The system oscillates autonomously with a near 24 h period, remains phase coherent for many days, and allows real-time observation of each component simultaneously without user intervention. This reassembled clock system provides new insights into how a circadian clock exerts control over gene expression and can serve in the area of synthetic biology as a new platform upon which to build even more complexity.One Sentence SummaryAn autonomously oscillating circadian clock-controlled gene regulatory circuit is studied in vitro using a real-time high-throughput assay.

In Salmonella enterica, 2-aminoacrylate (2AA) is a reactive enamine intermediate generated during a number of biochemical reactions. When the 2-iminobutanoate/2-iminopropanoate deaminase (RidA, EC: 3.5.99.10) is eliminated, 2AA accumulates and inhibits the activity of multiple pyridoxal 5&rsquo, phosphate(PLP)-dependent enzymes. In this study, untargeted proton nuclear magnetic resonance (1H NMR) metabolomics and transcriptomics data were used to uncover the global metabolic response of S. enterica to the accumulation of 2AA. The data showed that elimination of RidA perturbed folate and branched chain amino acid metabolism. Many of the resulting perturbations were consistent with the known effect of 2AA stress, while other results suggested additional potential enzyme targets of 2AA-dependent damage. The majority of transcriptional and metabolic changes appeared to be the consequence of downstream effects on the metabolic network, since they were not directly attributable to a PLP-dependent enzyme. In total, the results highlighted the complexity of changes stemming from multiple perturbations of the metabolic network, and suggested hypotheses that will be valuable in future studies of the RidA paradigm of endogenous 2AA stress.

Metabolic networks are webs of integrated reactions organized to maximize growth and replication while minimizing the detrimental impact that reactive metabolites can have on fitness. Enamines and imines, such as 2-aminoacrylate (2AA), are reactive metabolites produced as short-lived intermediates in a number of enzymatic processes. Left unchecked, the inherent reactivity of enamines and imines may perturb the metabolic network. Genetic and biochemical studies have outlined a role for the broadly conserved reactive intermediate deaminase (Rid) (YjgF/YER057c/UK114) protein family, in particular RidA, in catalyzing the hydrolysis of enamines and imines to their ketone product. Herein, we discuss new findings regarding the biological significance of enamine and imine production and outline the importance of RidA in controlling the accumulation of reactive metabolites.

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.

Reactive enamine stress caused by intracellular 2-aminoacrylate accumulation leads to pleiotropic growth defects in a variety of organisms. Members of the well-conserved RidA/YER057c/UK114 protein family prevent enamine stress by enhancing the breakdown of 2-aminoacrylate to pyruvate. In Salmonella enterica , disruption of RidA allows 2-aminoacrylate to accumulate and to inactivate a variety of pyridoxal 5′-phosphate-dependent enzymes by generating covalent bonds with the enzyme and/or cofactor. This study was initiated to identify mechanisms that can overcome 2-aminoacrylate stress in the absence of RidA. Multicopy suppressor analysis revealed that overproduction of the methionine biosynthesis enzyme cystathionine β-lyase (MetC) (EC 4.4.1.8) alleviated the pleiotropic consequences of 2-aminoacrylate stress in a ridA mutant strain. Degradation of cystathionine by MetC was not required for suppression of ridA phenotypes. The data support a model in which MetC acts on a noncystathionine substrate to generate a metabolite that reduces 2-aminoacrylate levels, representing a nonenzymatic mechanism of 2-aminoacrylate depletion. IMPORTANCE RidA proteins are broadly conserved and have been demonstrated to deaminate 2-aminoacrylate and other enamines. 2-Aminoacrylate is generated as an obligatory intermediate in several pyridoxal 5′-phosphate-dependent reactions; if it accumulates, it damages cellular enzymes. This study identified a novel mechanism to eliminate 2-aminoacrylate stress that required the overproduction, but not the canonical activity, of cystathionine β-lyase. The data suggest that a metabolite-metabolite interaction is responsible for quenching 2-aminoacrylate, and they emphasize the need for emerging technologies to probe metabolism in vivo .

Coenzyme A (CoA) is a ubiquitous coenzyme involved in fundamental metabolic processes. CoA is synthesized from pantothenic acid by a pathway that is largely conserved among bacteria and eukaryotes and consists of five enzymatic steps. While higher organisms, including humans, must scavenge pantothenate from the environment, most bacteria and plants are capable of de novo pantothenate biosynthesis. In Salmonella enterica , precursors to pantothenate can be salvaged, but subsequent intermediates are not transported due to their phosphorylated state, and thus the pathway from pantothenate to CoA is considered essential. Genetic analyses identified the STM4195 gene product of Salmonella enterica serovar Typhimurium as a transporter of pantothenate precursors, ketopantoate and pantoate and, to a lesser extent, pantothenate. Further results indicated that STM4195 transports a product of CoA degradation that serves as a precursor to CoA and enters the biosynthetic pathway between PanC and CoaBC ( dfp ). The relevant CoA derivative is distinguishable from pantothenate, pantetheine, and pantethine and has spectral properties indicating the adenine moiety of CoA is intact. Taken together, the results presented here provide evidence of a transport mechanism for the uptake of ketopantoate, pantoate, and pantothenate and demonstrate a role for STM4195 in the salvage of a CoA derivative of unknown structure. The STM4195 gene is renamed panS to reflect participation in pan tothenate s alvage that was uncovered herein. IMPORTANCE This manuscript describes a transporter for two pantothenate precursors in addition to the existence and transport of a salvageable coenzyme A (CoA) derivative. Specifically, these studies defined a function for an STM protein in S. enterica that was distinct from the annotated role and led to its designation as PanS ( pan tothenate s alvage). The presence of a salvageable CoA derivative and a transporter for it suggests the possibility that this compound is present in the environment and may serve a role in CoA synthesis for some organisms. As such, this work raises important question about CoA salvage that can be pursued with future studies in bacteria and other organisms.

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. 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.

Microorganisms respond to a variety of metabolic perturbations by repurposing or recruiting pathways to reroute metabolic flux and overcome the perturbation. Elimination of the 2-dehydropantoate 2-reductase, PanE, both reduces total coenzyme A (CoA) levels and causes a conditional HMP-P auxotrophy in Salmonella enterica. CoA or acetyl-CoA has no demonstrable effect on the HMP-P synthase, ThiC, in vitro. Suppressors aimed at probing the connection between the biosynthesis of thiamine and CoA contained mutations in the gene encoding the ilvC transcriptional regulator, ilvY. These mutations may help inform the structure and mechanism of action for the effector-binding domain, as they represent the first sequenced substitutions in the effector-binding domain of IlvY that cause constitutive expression of ilvC. Since IlvC moonlights as a 2-dehydropantoate 2-reductase, the resultant increase in ilvC transcription increased synthesis of CoA. This study failed to identify mutations overcoming the need for CoA for thiamine synthesis in S. enterica panE mutants, suggesting that a more integrated approach may be necessary to uncover the mechanism connecting CoA and ThiC activity in vivo.

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.

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 crystal structures of SPO0140 and Sbal_2486 were determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). The structures revealed a conserved core with domain duplication and a superficial similarity of the C-terminal domain to pleckstrin homology-like folds. The conservation of the domain interface indicates a potential binding site that is likely to involve a nucleotide-based ligand, with genome-context and gene-fusion analyses additionally supporting a role for this family in signal transduction, possibly during oxidative stress.

The crystal structure of the NGO1945 gene product from N. gonorrhoeae (UniProt Q5F5IO) reveals that the N-terminal domain assigned as a domain of unknown function (DUF2063) is likely to bind DNA and that the protein may be involved in transcriptional regulation., Proteins with the DUF2063 domain constitute a new Pfam family, PF09836. The crystal structure of a member of this family, NGO1945 from Neisseria gonorrhoeae, has been determined and reveals that the N-terminal DUF2063 domain is likely to be a DNA-binding domain. In conjunction with the rest of the protein, NGO1945 is likely to be involved in transcriptional regulation, which is consistent with genomic neighborhood analysis. Of the 216 currently known proteins that contain a DUF2063 domain, the most significant sequence homologs of NGO1945 (∼40–99% sequence identity) are from various Neisseria and Haemophilus species. As these are important human pathogens, NGO1945 represents an interesting candidate for further exploration via biochemical studies and possible therapeutic intervention.

KPN03535 is a protein unique to K. pneumoniae. The crystal structure reveals that KPN03535 represents a novel variant of the OB-fold and is likely to be a DNA-binding lipoprotein., KPN03535 (gi|152972051) is a putative lipoprotein of unknown function that is secreted by Klebsiella pneumoniae MGH 78578. The crystal structure reveals that despite a lack of any detectable sequence similarity to known structures, it is a novel variant of the OB-fold and structurally similar to the bacterial Cpx-pathway protein NlpE, single-stranded DNA-binding (SSB) proteins and toxins. K. pneumoniae MGH 78578 forms part of the normal human skin, mouth and gut flora and is an opportunistic pathogen that is linked to about 8% of all hospital-acquired infections in the USA. This structure provides the foundation for further investigations into this divergent member of the OB-fold family.

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.

The protein SO4266 (gi|24375750) from the bacterium Shewanella oneidensis MR-1 is annotated as a member of Pfam PF02661. This family consists of Fic (filamentation induced by cAMP) proteins and their relatives, and is characterized by the presence of a well-conserved HPFXXGNG motif 1. The biochemistry of Fic proteins has not been characterized extensively and their exact molecular functions remain unknown. From early studies in Escherichia coli, it is believed that Fic proteins and cAMP may be involved in a regulatory mechanism of cell division, including folate metabolism by the synthesis of p-aminobenzoic acid (PABA) or folate 1. Proteins containing the Fic domain are present in all kingdoms of life and range in size from ~200 to 500 amino acids. The Fic protein family contains 647 members, including two human proteins, according to Pfam (May 2008). Sequence-based clustering 2 of this protein family, at 30% sequence identity, groups these proteins into 18 clusters. Three crystal structures of Fic proteins from bacteria (unpublished) are available in the Protein Data Bank [accession codes 2g03 (194 residues, 2.2 A), 2f6s (201 residues, 2.5 A) and 3cuc (262 residues, 2.7 A)]. The first two of these proteins belong to a single cluster of 16 members and share ~60% sequence identity. The anti-apoptotic bacterial effector protein BepA, which is a type IV secretion (T4S) system substrate, also contains an N-terminal Fic domain 3. In humans, the Fic domain is present in the Huntingtin Interacting Protein E (HYPE; Uniprot entry Q9BVA6_HUMAN), a protein of unknown function that is thought to interact with Huntingtin, one of the major proteins in the Huntington's disease protein interaction network (listed as NAD- or FAD-binding) 4. Bioinformatics analysis of prokaryotic toxin-antitoxin networks 5 suggests that Fic proteins are putative death-on-curing (Doc) toxins that are part of the Phd-Doc system. These proteins likely function as metal-dependent nucleases or RNA-processing enzymes, 5 while more recent studies suggest that Doc toxicity is caused by inhibition of translation elongation 6. SO4266 (Uniprot entry Q8E9K5_SHEON), at 372 amino acids, is one of the largest Fic domain-containing proteins to have its structure determined. Interestingly, both HYPE and SO4266 belong to the largest sequence cluster in this family (n.b. our B. thetaiotaomicron {"type":"entrez-protein","attrs":{"text":"NP_811426.1","term_id":"29347923","term_text":"NP_811426.1"}}NP_811426.1 structure with PDB id 3cuc also belongs to this cluster), which comprises 466 out of 647 proteins, and share ~32% sequence identity in the Fic domain. Here, we report the crystal structure of the SO4266 protein at 1.6 A resolution. The structure reveals a dimeric protein with additional electron density in the vicinity of the highly conserved HPFXXGNG motif in the Fic domain of one subunit that corresponds to the N-terminus of a symmetry-related molecule. In addition, the study also reveals a C-terminal winged-helix DNA-binding domain that sets it apart from the other Fic protein structures. The structure presented here is a representative of the largest sequence cluster and together with the structures of the other Fic proteins paves the way for further structure-based functional characterization.

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 (PLP)-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.

RidA, the archetype member of the widely conserved RidA/YER057c/UK114 family of proteins, prevents reactive enamine/imine intermediates from accumulating in Salmonella enterica by catalyzing their hydrolysis to stable keto acid products. In the absence of RidA, endogenous 2-aminoacrylate persists in the cellular environment long enough to damage a growing list of essential metabolic enzymes. Prior studies have focused on the dehydration of serine by the pyridoxal 5′-phosphate (PLP)-dependent serine/threonine dehydratases, IlvA and TdcB, as sources of endogenous 2-aminoacrylate. The current study describes an additional source of endogenous 2-aminoacrylate derived from cysteine. The results of in vivo analysis show that the cysteine sensitivity of a ridA strain is contingent upon CdsH, the predominant cysteine desulfhydrase in S. enterica . The impact of cysteine on 2-aminoacrylate accumulation is shown to be unaffected by the presence of serine/threonine dehydratases, revealing another mechanism of endogenous 2-aminoacrylate production. Experiments in vitro suggest that 2-aminoacrylate is released from CdsH following cysteine desulfhydration, resulting in an unbound aminoacrylate substrate for RidA. This work expands our understanding of the role played by RidA in preventing enamine stress resulting from multiple normal metabolic processes.

Coenzyme A (CoA) is an essential cofactor required in all living organisms, where it functions primarily as an acyl-group carrier and activator of carbonyl groups. The biosynthetic pathway generati...

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., Proteins that contain the DUF2874 domain constitute a new Pfam family PF11396. Members of this family have predominantly been identified in microbes found in the human gut and oral cavity. The crystal structure of one member of this family, BVU2987 from Bacteroides vulgatus, has been determined, revealing a β-lactamase inhibitor protein-like structure with a tandem repeat of domains. Sequence analysis and structural comparisons reveal that BVU2987 and other DUF2874 proteins are related to β-lactamase inhibitor protein, PepSY and SmpA_OmlA proteins and hence are likely to function as inhibitory proteins.

Structures of the first representatives of PF06684 (DUF1185) reveal a Bacillus chorismate mutase-like fold with a potential role in amino-acid synthesis., The crystal structures of BB2672 and SPO0826 were determined to resolutions of 1.7 and 2.1 Å by single-wavelength anomalous dispersion and multiple-wavelength anomalous dispersion, respectively, using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG) as part of the NIGMS Protein Structure Initiative (PSI). These proteins are the first structural representatives of the PF06684 (DUF1185) Pfam family. Structural analysis revealed that both structures adopt a variant of the Bacillus chorismate mutase fold (BCM). The biological unit of both proteins is a hexamer and analysis of homologs indicates that the oligomer interface residues are highly conserved. The conformation of the critical regions for oligomerization appears to be dependent on pH or salt concentration, suggesting that this protein might be subject to environmental regulation. Structural similarities to BCM and genome-context analysis suggest a function in amino-acid synthesis.

The crystal structure of a putative NTP pyrophosphohydrolase, YP_001813558.1 from E. sibiricum, reveals a novel segment-swapped linked-dimer assembly., The crystal structure of a putative NTPase, YP_001813558.1 from Exiguo­bacterium sibiricum 255-15 (PF09934, DUF2166) was determined to 1.78 Å resolution. YP_001813558.1 and its homologs (dimeric dUTPases, MazG proteins and HisE-encoded phosphoribosyl ATP pyrophosphohydrolases) form a superfamily of all-α-helical NTP pyrophosphatases. In dimeric dUTPase-like proteins, a central four-helix bundle forms the active site. However, in YP_001813558.1, an unexpected intertwined swapping of two of the helices that compose the conserved helix bundle results in a ‘linked dimer’ that has not previously been observed for this family. Interestingly, despite this novel mode of dimerization, the metal-binding site for divalent cations, such as magnesium, that are essential for NTPase activity is still conserved. Furthermore, the active-site residues that are involved in sugar binding of the NTPs are also conserved when compared with other α-helical NTPases, but those that recognize the nucleotide bases are not conserved, suggesting a different substrate specificity.

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.

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.