Summary

Gluconeogenesis converts three carbon units into glucose. Here we identify an analogous pathway in Saccharomyces cerevisiae for converting three carbon units into ribose, a component of nucleic acids and nucleotides. This riboneogenic pathway involves the enzyme sedoheptulose-1,7-bisphosphatase (SHB17), whose activity was identified based on accumulation of sedoheptulose-1,7-bisphosphate in the corresponding knockout strain. We determined the crystal structure of Shb17 in complex with sedoheptulose-1,7-bisphosphate, and found that the sugar is bound in the closed furan form in the active site. Like fructose-1,6-bisphosphate, sedoheptulose-1,7-bisphosphate is produced by aldolase, in this case from erythrose 4-phosphate and dihydroxyacetone phosphate. Hydrolysis of sedoheptulose-1,7-bisphosphate by SHB17 provides an energetically favorable input to the non-oxidative pentose phosphate pathway to drive ribose production. Flux through SHB17 is enhanced under conditions when ribose demand is high relative to demand for NADPH, including during ribosome biogenesis in metabolically synchronized yeast cells. Thus, riboneogenesis provides a thermodynamically-driven route of ribose production uncoupled from formation of NADPH.

The carbon-fluorine bond is the strongest covalent bond in organic chemistry, yet fluoroacetate dehalogenases can readily hydrolyze this bond under mild physiological conditions. Elucidating the molecular basis of this rare biocatalytic activity will provide the fundamental chemical insights of how this formidable feat is achieved. Here, we present a series of high-resolution (1.15–1.80 Å) crystal structures of a fluoroacetate dehalogenase, capturing snapshots along the defluorination reaction: the free enzyme, enzyme-fluoroacetate Michaelis complex, glycolyl-enzyme covalent intermediate and enzyme-product complex. We demonstrate that enzymatic defluorination requires a halide pocket that not only supplies three hydrogen bonds to stabilize the fluoride ion, but is also finely tailored for the smaller fluorine halogen atom to establish selectivity towards fluorinated substrates. We have further uncovered dynamics near the active site which may play pivotal roles in enzymatic defluorination. These findings may ultimately lead to the development of novel defluorinases that will enable the biotransformation of more complex fluorinated organic compounds, which in turn will assist the synthesis, detoxification, biodegradation, disposal, recycling and regulatory strategies for the growing markets of organofluorines across major industrial sectors.

Summary

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks, and 5′-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.

Cyclic diguanylate (c-di-GMP) is a ubiquitous second messenger regulating diverse cellular functions including motility, biofilm formation, cell cycle progression and virulence in bacteria. In the cell, degradation of c-di-GMP is catalyzed by highly specific EAL domain phosphodiesterases whose catalytic mechanism is still unclear. Here, we purified 13 EAL domain proteins from various organisms and demonstrated that their catalytic activity is associated with the presence of 10 conserved EAL domain residues. The crystal structure of the TDB1265 EAL domain was determined in a free state (1.8 Å) and in complex with c-di-GMP (2.35 Å) and unveiled the role of the conserved residues in substrate binding and catalysis. The structure revealed the presence of two metal ions directly coordinated by six conserved residues, two oxygens of the c-di-GMP phosphate, and potential catalytic water molecule. Our results support a two-metal-ion catalytic mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.

Type II chromosomal toxin-antitoxin (TA) modules consist of a pair of genes that encode two components: a stable toxin and a labile antitoxin interfering with the lethal action of the toxin through protein complex formation. Bioinformatic analysis of Streptococcus mutans UA159 genome identified a pair of linked genes encoding a MazEF-like TA. Our results show that S. mutans mazEF genes form a bicistronic operon that is cotranscribed from a σ70-like promoter. Overproduction of S. mutans MazF toxin had a toxic effect on S. mutans which can be neutralized by coexpression of its cognate antitoxin, S. mutans MazE. Although mazF expression inhibited cell growth, no cell lysis of S. mutans cultures was observed under the conditions tested. The MazEF TA is also functional in E. coli, where S. mutans MazF did not kill the cells but rather caused reversible cell growth arrest. Recombinant S. mutans MazE and MazF proteins were purified and were shown to interact with each other in vivo, confirming the nature of this TA as a type II addiction system. Our data indicate that MazF is a toxic nuclease arresting cell growth through the mechanism of RNA cleavage and that MazE inhibits the RNase activity of MazF by forming a complex. Our results suggest that the MazEF TA module might represent a cell growth modulator facilitating the persistence of S. mutans under the harsh conditions of the oral cavity.

The genome of the extremely radiation resistant bacterium Deinococcus radiodurans encodes 21 Nudix hydrolases of which only two have been characterized in detail. Here we report the activity and crystal structure for DR_0079, the first Nudix hydrolase observed to have a marked preference for cytosine ribonucleoside 5’-diphosphate (CDP) and cytosine ribonucleoside 5’-triphosphate (CTP). After CDP and CTP the next most preferred substrates for DR_0079, with a relative activity of < 50%, were the corresponding deoxyribose nucleotides, dCDP and dCTP. Hydrolase activity at the site of the phosphodiester bond was corroborated using 31P NMR spectroscopy to follow the phosphorus resonances for three substrates, CDP, IDP, and CTP, and their respective hydrolysis products, CMP + Pi, IMP + Pi, and CMP + PPi. Nucleophilic substitution at the β-phosphorus of CDP and CTP was established, using 31P NMR spectroscopy, by the appearance of an upfield shifted Pi resonance and line-broadened PPi resonance, respectively, when performing the hydrolysis in 40% H218O enriched water. Optimum activity for CDP was at pH 9.0 – 9.5 with the reaction requiring divalent metal cation (Mg2+ > Mn2+ > Co2+). The biochemical data is discussed with reference to the crystal structure for DR_0079 that was determined in the metal-free form at 1.9 Å resolution. The protein contains nine β-strands, three α-helices, and two 310-helices organized into three subdomains; an N-terminal β-sheet, a central Nudix core, and a C-terminal helix-turn-helix motif. As observed for all known structures of Nudix hydrolases, the α-helix of the ‘Nudix box’ is one of two helices that sandwich a ‘four-strand’ mixed β-sheet. To identify residues potentially involved in metal and substrate binding, NMR chemical shift mapping experiments were performed on 15N-labelled DR_0079 with the paramagnetic divalent cation Co2+ and the non-hydrolyzable substrate thymidine-5’-O-(α,β-methylenediphosphate) and the results mapped onto the crystal structure.

The protein TA0175 has a large number of sequence homologues, most of which are annotated as unknown and a few as belonging to the haloacid dehalogenase superfamily, but has no known biological function. Using a combination of amino acid sequence analysis, three-dimensional crystal structure information, and kinetic analysis, we have characterized TA0175 as phosphoglycolate phosphatase from Thermoplasma acidophilum. The crystal structure of TA0175 revealed two distinct domains, a larger core domain and a smaller cap domain. The large domain is composed of a centrally located five-stranded parallel β-sheet with strand order S10, S9, S8, S1, S2 and a small β-hairpin, strands S3 and S4. This central sheet is flanked by a set of three α-helices on one side and two helices on the other. The smaller domain is composed of an open faced β-sandwich represented by three antiparallel β-strands, S5, S6, and S7, flanked by two oppositely oriented α-helices, H3 and H4. The topology of the large domain is conserved; however, structural variation is observed in the smaller domain among the different functional classes of the haloacid dehalogenase superfamily. Enzymatic assays on TA0175 revealed that this enzyme catalyzed the dephosphorylation of phosphoglycolate in vitro with similar kinetic properties seen for eukaryotic phosphoglycolate phosphatase. Activation by divalent cations, especially Mg2+, and competitive inhibition behavior with Cl− ions are similar between TA0175 and phosphoglycolate phosphatase. The experimental evidence presented for TA0175 is indicative of phosphoglycolate phosphatase.

Structural proteomics projects are generating three-dimensional structures of novel, uncharacterized proteins at an increasing rate. However, structure alone is often insufficient to deduce the specific biochemical function of a protein. Here we determined the function for a protein using a strategy that integrates structural and bioinformatics data with parallel experimental screening for enzymatic activity. BioH is involved in biotin biosynthesis in Escherichia coli and had no previously known biochemical function. The crystal structure of BioH was determined at 1.7 Å resolution. An automated procedure was used to compare the structure of BioH with structural templates from a variety of different enzyme active sites. This screen identified a catalytic triad (Ser82, His235, and Asp207) with a configuration similar to that of the catalytic triad of hydrolases. Analysis of BioH with a panel of hydrolase assays revealed a carboxylesterase activity with a preference for short acyl chain substrates. The combined use of structural bioinformatics with experimental screens for detecting enzyme activity could greatly enhance the rate at which function is determined from structure.

Glutaminases belong to the large superfamily of serine-dependent β-lactamases and penicillin-binding proteins, and they catalyze the hydrolytic deamidation of l-glutamine to l-glutamate. In this work, we purified and biochemically characterized four predicted glutaminases from Escherichia coli (YbaS and YneH) and Bacillus subtilis (YlaM and YbgJ). The proteins demonstrated strict specificity to l-glutamine and did not hydrolyze d-glutamine or l-asparagine. In each organism, one glutaminase showed higher affinity to glutamine (E. coli YbaS and B. subtilis YlaM; Km 7.3 and 7.6 mM, respectively) than the second glutaminase (E. coli YneH and B. subtilis YbgJ; Km 27.6 and 30.6 mM, respectively). The crystal structures of the E. coli YbaS and the B. subtilis YbgJ revealed the presence of a classical β-lactamase-like fold and conservation of several key catalytic residues of β-lactamases (Ser74, Lys77, Asn126, Lys268, and Ser269 in YbgJ). Alanine replacement mutagenesis demonstrated that most of the conserved residues located in the putative glutaminase catalytic site are essential for activity. The crystal structure of the YbgJ complex with the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine revealed the presence of a covalent bond between the inhibitor and the hydroxyl oxygen of Ser74, providing evidence that Ser74 is the primary catalytic nucleophile and that the glutaminase reaction proceeds through formation of an enzyme–glutamyl intermediate. Growth experiments with the E. coli glutaminase deletion strains revealed that YneH is involved in the assimilation of l-glutamine as a sole source of carbon and nitrogen and suggested that both glutaminases (YbaS and YneH) also contribute to acid resistance in E. coli.

We have identified a novel family of proteins, in which the N-terminal Cystathionine Beta-Synthase (CBS) domain is fused to the C-terminal Zn ribbon domain. Four proteins were over-expressed in E. coli and purified: TA0289 from Thermoplasma acidophilum, TV1335 from Thermoplasma vulcanum, PF1953 from Pyrococcus furiosus, and PH0267 from Pyrococcus horikoshii. The purified proteins had red/purple color in solution and an absorption spectrum typical of rubredoxins. Metal analysis of purified proteins revealed the presence of several metals with iron and zinc being the most abundant metals (2 to 67% of iron and 12 to 74% of zinc). Crystal structures of both mercury- and iron-bound TA0289 (1.5–2.0 Å resolution) revealed a dimeric protein whose inter-subunit contacts are formed exclusively by the α helices of two CBS sub-domains, whereas the C-terminal domain has a classical Zn-ribbon planar architecture. All proteins were reversibly reduced by chemical reductants (ascorbate or dithionite) or by the general rubredoxin reductase NorW from E. coli in the presence of NADH. Reduced TA0289 was found to be able to transfer electrons to cytochrome C from horse heart. Likewise, the purified Zn ribbon protein KTI11 from Saccharomyces cerevisiae had purple color in solution and a rubredoxin-like absorption spectrum, contained both iron and zinc, and was reduced by the rubredoxin reductase NorW from E. coli. Thus, recombinant Zn ribbon domains from archaea and yeast demonstrate a rubredoxin-like electron carrier activity in vitro. We suggest that in vivo some Zn ribbon domains might also bind iron and therefore possess an electron carrier activity adding another physiological role to this large family of important proteins.

Rhodobacter capsulatus possesses two genes potentially coding for ammonia transporters, amtB and amtY. In order to better understand their role in the physiology of this bacterium and their possible significance in nitrogen fixation, we created single-knockout mutants. Strains mutated in either amtB or amtY did not show a growth defect under any condition tested and were still capable of taking up ammonia at nearly wild-type rates, but an amtB mutant was no longer capable of transporting methylamine. The amtB strain but not the amtY strain was also totally defective in carrying out ADP-ribosylation of Fe-protein or the switch-off of in vivo nitrogenase activity in response to NH4+ addition. ADP-ribosylation in response to darkness was unaffected in amtB and amtBY strains, and glutamine synthetase activity was normally regulated in these strains in response to ammonium addition, suggesting that one role of AmtB is to function as an ammonia sensor for the processes that regulate nitrogenase activity.

The photosynthetic bacterium Rhodobacter capsulatus has been shown to regulate its nitrogenase by covalent modification via the reversible ADP-ribosylation of Fe protein in response to darkness or the addition of external NH4+. Here we demonstrate the presence of ADP-ribosylated Fe protein under a variety of steady-state growth conditions. We examined the modification of Fe protein and nitrogenase activity under three different growth conditions that establish different levels of cellular nitrogen: batch growth with limiting NH4+, where the nitrogen status is externally controlled; batch growth on relatively poor nitrogen sources, where the nitrogen status is internally controlled by assimilatory processes; and continuous culture. When cultures were grown to stationary phase with different limiting concentrations of NH4+, the ADP-ribosylation state of Fe protein was found to correlate with cellular nitrogen status. Additionally, actively growing cultures (grown with N2 or glutamate), which had an intermediate cellular nitrogen status, contained a portion of their Fe protein in the modified state. The correlation between cellular nitrogen status and ADP-ribosylation state was corroborated with continuous cultures grown under various degrees of nitrogen limitation. These results show that in R. capsulatus the modification system that ADP-ribosylates nitrogenase in the short term in response to abrupt changes in the environment is also capable of modifying nitrogenase in accordance with long-term cellular conditions.

The photosynthetic bacterium Rhodobacter capsulatus has been shown to carry out nitrogenase “switch-off,” a rapid, reversible inhibition of in vivo activity. Here, we demonstrate that highly nitrogen-limited cultures of both the wild-type strain and a draT draG mutant are capable of nitrogenase switch-off while moderately nitrogen-limited cultures show instead a “magnitude” response, with a decrease in in vivo nitrogenase activity that is proportional to the amount of added NH4+.

The CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of evolution for both the cas genes and the unique spacer content. Here, we provide an updated analysis of the evolutionary relationships between CRISPR–Cas systems and Cas proteins. Three major types of CRISPR–Cas system are delineated, with a further division into several subtypes and a few chimeric variants. Given the complexity of the genomic architectures and the extremely dynamic evolution of the CRISPR–Cas systems, a unified classification of these systems should be based on multiple criteria. Accordingly, we propose a `polythetic' classification that integrates the phylogenies of the most common cas genes, the sequence and organization of the CRISPR repeats and the architecture of the CRISPR–cas loci.