γδ T cells exhibit multifunctional and protective memory in intestinal tissues.

The study of T cell memory and the target of vaccine design have focused on memory subsumed by T cells bearing the αß T cell receptor. Alternatively, γδ T cells are thought to provide rapid immunity, particularly at mucosal borders. Here, we have shown that a distinct subset of mucosal γδ T cells mounts an immune response to oral Listeria monocytogenes (Lm) infection and leads to the development of multifunctional memory T cells capable of simultaneously producing interferon-γ and interleukin-17A in the murine intestinal mucosa. Challenge infection with oral Lm, but not oral Salmonella or intravenous Lm, induced rapid expansion of memory γδ T cells, suggesting contextual specificity to the priming pathogen. Importantly, memory γδ T cells were able to provide enhanced protection against infection. These findings illustrate that γδ T cells play a role with hallmarks of adaptive immunity in the intestinal mucosa.

Characterization and homology modelling of a novel multi-modular and multi-functional Paenibacillus mucilaginosus glycoside hydrolase.

Glycoside hydrolases, particularly cellulases, xylanases and mannanases, are essential for the depolymerisation of lignocellulosic substrates in various industrial bio-processes. In the present study, a novel glycoside hydrolase from Paenibacillus mucilaginosus (PmGH) was expressed in E. coli, purified and characterised. Functional analysis indicated that PmGH is a 130 kDa thermophilic multi-modular and multi-functional enzyme, comprising a GH5, a GH6 and two CBM3 domains and exhibiting cellulase, mannanase and xylanase activities. The enzyme displayed optimum hydrolytic activities at pH 6 and 60 °C and moderate thermostability. Homology modelling of the full-length protein highlighted the structural and functional novelty of native PmGH, with no close structural homologs identified. However, homology modelling of the individual GH5, GH6 and the two CBM3 domains yielded excellent models based on related structures from the Protein Data Bank. The catalytic GH5 and GH6 domains displayed a (ß/α)8 and a distorted seven stranded (ß/α) fold, respectively. The distinct homology at the domain level but low homology of the full-length protein suggests that this protein evolved by exogenous gene acquisition and recombination.

Structural Characterization and Directed Evolution of a Novel Acetyl Xylan Esterase Reveals Thermostability Determinants of the Carbohydrate Esterase 7 Family.

A hot desert hypolith metagenomic DNA sequence data set was screened in silico for genes annotated as acetyl xylan esterases (AcXEs). One of the genes identified encoded an ∼36-kDa protein (Axe1NaM1). The synthesized gene was cloned and expressed, and the resulting protein was purified. NaM1 was optimally active at pH 8.5 and 30°C and functionally stable at salt concentrations of up to 5 M. The specific activity and catalytic efficiency were 488.9 U mg-1 and 3.26 × 106 M-1 s-1, respectively. The crystal structure of wild-type NaM1 was solved at a resolution of 2.03 Å, and a comparison with the structures and models of more thermostable carbohydrate esterase 7 (CE7) family enzymes and variants of NaM1 from a directed evolution experiment suggests that reduced side-chain volume of protein core residues is relevant to the thermal stability of NaM1. Surprisingly, a single point mutation (N96S) not only resulted in a simultaneous improvement in thermal stability and catalytic efficiency but also increased the acyl moiety substrate range of NaM1.IMPORTANCE AcXEs belong to nine carbohydrate esterase families (CE1 to CE7, CE12, and CE16), of which CE7 enzymes possess a unique and narrow specificity for acetylated substrates. All structurally characterized members of this family are moderately to highly thermostable. The crystal structure of a novel, mesophilic CE7 AcXE (Axe1NaM1), from a soil metagenome, provides a basis for comparisons with thermostable CE7 enzymes. Using error-prone PCR and site-directed mutagenesis, we enhanced both the stability and activity of the mesophilic AcXE. With comparative structural analyses, we have also identified possible thermal stability determinants. These are valuable for understanding the thermal stability of enzymes within this family and as a guide for future protein engineering of CE7 and other α/ß hydrolase enzymes.

Resistance related metabolic pathways for drug target identification in Mycobacterium tuberculosis.

BACKGROUND: Increasing resistance to anti-tuberculosis drugs has driven the need for developing new drugs. Resources such as the tropical disease research (TDR) target database and AssessDrugTarget can help to prioritize putative drug targets. Hower, these resources do not necessarily map to metabolic pathways and the targets are not involved in dormancy. In this study, we specifically identify drug resistance pathways to allow known drug resistant mutations in one target to be offset by inhibiting another enzyme of the same metabolic pathway. One of the putative targets, Rv1712, was analysed by modelling its three dimensional structure and docking potential inhibitors. RESULTS: We mapped 18 TB drug resistance gene products to 15 metabolic pathways critical for mycobacterial growth and latent TB by screening publicly available microarray data. Nine putative targets, Rv1712, Rv2984, Rv2194, Rv1311, Rv1305, Rv2195, Rv1622c, Rv1456c and Rv2421c, were found to be essential, to lack a close human homolog, and to share >67 % sequence identity and >87 % query coverage with mycobacterial orthologs. A structural model was generated for Rv1712, subjected to molecular dynamic simulation, and identified 10 compounds with affinities better than that for the ligand cytidine-5'-monophosphate (C5P). Each compound formed more interactions with the protein than C5P. CONCLUSIONS: We focused on metabolic pathways associated with bacterial drug resistance and proteins unique to pathogenic bacteria to identify novel putative drug targets. The ten compounds identified in this study should be considered for experimental studies to validate their potential as inhibitors of Rv1712.

Structure of ADP-aluminium fluoride-stabilized protochlorophyllide oxidoreductase complex.

Photosynthesis uses chlorophylls for the conversion of light into chemical energy, the driving force of life on Earth. During chlorophyll biosynthesis in photosynthetic bacteria, cyanobacteria, green algae and gymnosperms, dark-operative protochlorophyllide oxidoreductase (DPOR), a nitrogenase-like metalloenzyme, catalyzes the chemically challenging two-electron reduction of the fully conjugated ring system of protochlorophyllide a. The reduction of the C-17=C-18 double bond results in the characteristic ring architecture of all chlorophylls, thereby altering the absorption properties of the molecule and providing the basis for light-capturing and energy-transduction processes of photosynthesis. We report the X-ray crystallographic structure of the substrate-bound, ADP-aluminium fluoride-stabilized (ADP·AlF(3)-stabilized) transition state complex between the DPOR components L(2) and (NB)(2) from the marine cyanobacterium Prochlorococcus marinus. Our analysis permits a thorough investigation of the dynamic interplay between L(2) and (NB)(2). Upon complex formation, substantial ATP-dependent conformational rearrangements of L(2) trigger the protein-protein interactions with (NB)(2) as well as the electron transduction via redox-active [4Fe-4S] clusters. We also present the identification of artificial "small-molecule substrates" of DPOR in correlation with those of nitrogenase. The catalytic differences and similarities between DPOR and nitrogenase have broad implications for the energy transduction mechanism of related multiprotein complexes that are involved in the reduction of chemically stable double and/or triple bonds.

Structure and functional characterization of pyruvate decarboxylase from Gluconacetobacter diazotrophicus.

BACKGROUND: Bacterial pyruvate decarboxylases (PDC) are rare. Their role in ethanol production and in bacterially mediated ethanologenic processes has, however, ensured a continued and growing interest. PDCs from Zymomonas mobilis (ZmPDC), Zymobacter palmae (ZpPDC) and Sarcina ventriculi (SvPDC) have been characterized and ZmPDC has been produced successfully in a range of heterologous hosts. PDCs from the Acetobacteraceae and their role in metabolism have not been characterized to the same extent. Examples include Gluconobacter oxydans (GoPDC), G. diazotrophicus (GdPDC) and Acetobacter pasteutrianus (ApPDC). All of these organisms are of commercial importance. RESULTS: This study reports the kinetic characterization and the crystal structure of a PDC from Gluconacetobacter diazotrophicus (GdPDC). Enzyme kinetic analysis indicates a high affinity for pyruvate (K M 0.06 mM at pH 5), high catalytic efficiencies (1.3 • 10(6) M(-1) • s(-1) at pH 5), pHopt of 5.5 and Topt at 45°C. The enzyme is not thermostable (T½ of 18 minutes at 60°C) and the calculated number of bonds between monomers and dimers do not give clear indications for the relatively lower thermostability compared to other PDCs. The structure is highly similar to those described for Z. mobilis (ZmPDC) and A. pasteurianus PDC (ApPDC) with a rmsd value of 0.57 Å for Cα when comparing GdPDC to that of ApPDC. Indole-3-pyruvate does not serve as a substrate for the enzyme. Structural differences occur in two loci, involving the regions Thr341 to Thr352 and Asn499 to Asp503. CONCLUSIONS: This is the first study of the PDC from G. diazotrophicus (PAL5) and lays the groundwork for future research into its role in this endosymbiont. The crystal structure of GdPDC indicates the enzyme to be evolutionarily closely related to homologues from Z. mobilis and A. pasteurianus and suggests strong selective pressure to keep the enzyme characteristics in a narrow range. The pH optimum together with reduced thermostability likely reflect the host organisms niche and conditions under which these properties have been naturally selected for. The lack of activity on indole-3-pyruvate excludes this decarboxylase as the enzyme responsible for indole acetic acid production in G. diazotrophicus.

Structural and biophysical characterization of the secreted, ß-helical adhesin EtpA of Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a diarrhoeal pathogen associated with high morbidity and mortality especially among young children in developing countries. At present, there is no vaccine for ETEC. One candidate vaccine antigen, EtpA, is a conserved secreted adhesin that binds to the tips of flagellae to bridge ETEC to host intestinal glycans. EtpA is exported through a Gram-negative, two-partner secretion system (TPSS, type Vb) comprised of the secreted EtpA passenger (TpsA) protein and EtpB (TpsB) transporter that is integrated into the outer bacterial membrane. TpsA proteins share a conserved, N-terminal TPS domain followed by an extensive C-terminal domain with divergent sequence repeats. Two soluble, N-terminal constructs of EtpA were prepared and analysed respectively including residues 67 to 447 (EtpA67-447) and 1 to 606 (EtpA1-606). The crystal structure of EtpA67-447 solved at 1.76 Å resolution revealed a right-handed parallel ß-helix with two extra-helical hairpins and an N-terminal ß-strand cap. Analyses by circular dichroism spectroscopy confirmed the ß-helical fold and indicated high resistance to chemical and thermal denaturation as well as rapid refolding. A theoretical AlphaFold model of full-length EtpA largely concurs with the crystal structure adding an extended ß-helical C-terminal domain after an interdomain kink. We propose that robust folding of the TPS domain upon secretion provides a template to extend the N-terminal ß-helix into the C-terminal domains of TpsA proteins.

Structural and biophysical characterization of the multidomain xylanase Xyl.

The depletion of fossil fuels, associated pollution, and resulting health hazards are of concern worldwide. Woody biomass constitutes an alternative source of cleaner and renewable energy. The efficient use of woody biomass depends on xylan depolymerisation as the endo-ß-1,4-xylopyranosyl homopolymer is the main component of hemicellulose, the second most abundant component of wood. Xylan depolymerisation is achieved by hemicellulolytic xylanases of glycoside hydrolase (GH) families 5, 8, 10, 11, 30 and 43 of the CAZY database. We analysed a multidomain xylanase (Xyl) from the hindgut metagenome of the snouted harvester termite Trinervitermes trinervoides that releases xylobiose and xylotriose from beech and birch xylan and wheat arabinoxylan. The four domains of Xyl include an N-terminal GH11 xylanase domain, two family 36-like carbohydrate-binding domains CBM36-1 and 2, and a C-terminal CE4 esterase domain. Previous analyses indicated that CBM36-1 deletion slightly increased GH11 catalysis at low pH whereas removal of both CBMs decreased xylanase activity at 60°C from 90 to 56%. Possible cooperativity between the domains suggested by these observations was explored. A crystal structure of the two-domain construct, GH11-CBM36-1, confirmed the structure of the GH11 domain whereas the CBM36-1 domain lacked electron density, possibly indicating a random orientation of the CBM36-1 domain around the GH11 domain. Isothermal titration calorimetry (ITC) experiments similarly did not indicate specific interactions between the individual domains of Xyl supporting a "beads-on-a-string" model for Xyl domains.

Elucidation of repeat motifs R1- and R2-related TRIOBP variants in autosomal recessive nonsyndromic hearing loss DFNB28 among indigenous South African individuals.

BACKGROUND: DFNB28, a recessively inherited nonsyndromic form of deafness in humans, is caused by mutations in the TRIOBP gene (MIM #609761) on chromosome 22q13. Its protein TRIOBP helps to tightly bundle F-actin filaments, forming a rootlet that penetrates through the cuticular plate into the cochlear hair cell body. Repeat motifs R1 and R2, located in exon 7 of the TRIOBP-5 isoform, are the actin-binding domains. Deletion of both repeat motifs R1 and R2 results in complete disruption of both actin-binding and bundling activities, whereas deletion of the R2 motif alone retains F-actin bundling ability in stereocilia rootlets. METHODS: Target sequencing, using a custom capture panel of 180 known and candidate genes associated with sensorineural hearing loss, bioinformatics processing, and data analysis were performed. Genesis 2.0 was used for variant filtering based on quality/score read depth and minor allele frequency (MAF) thresholds of 0.005 for recessive NSHL, as reported in population-based sequencing databases. All variants were reclassified based on the American College of Medical Genetics and Genomics (ACMG) and Association for Molecular Pathology (AMP) guidelines together with other variant interpretation guidelines for genetic hearing loss . Candidate variants were confirmed via Sanger sequencing according to standard protocols, using the ABIPRISM 3730 DNA Analyzer. DNA sequence analysis was performed with DNASTAR Lasergene software. RESULTS: Candidate TRIOBP variants identified among 94 indigenous sub-Saharan African individuals were characterized through segregation analysis. Family TS005 carrying variants c.572delC, p.Pro191Argfs*50, and c.3510_3513dupTGCA, p.Pro1172Cysfs*13, demonstrated perfect cosegregation with the deafness phenotype. On the other hand, variants c.505C > A p.Asp168Glu and c.3636 T > A p.Leu1212Gln in the same family did not segregate with deafness and we have classified these variants as benign. A control family, TS067, carrying variants c.2532G > T p.Leu844Arg, c.2590C > A p.Asn867Lys, c.3484C > T p.Pro1161Leu, and c.3621 T > C p.Phe1187Leu demonstrated no cosegregation allowing us to classify these variants as benign. Together with published TRIOBP variants, the results showed that genotypes combining two truncating TRIOBP variants affecting repeat motifs R1 and R2 or R2 alone lead to a deafness phenotype, while a truncating variant affecting repeat motifs R1 and R2 or R2 alone combined with a missense variant does not. Homozygous truncating variants affecting repeat motif R2 cosegregate with the deafness phenotype. CONCLUSION: While a single intact R1 motif may be adequate for actin-binding and bundling in the stereocilia of cochlear hair cells, our findings indicate that a truncated R2 motif in cis seems to be incompatible with normal hearing, either by interfering with the function of an intact R1 motif or through another as yet unknown mechanism. Our study also suggests that most heterozygous missense variants involving exon 7 are likely to be tolerated.

Crystal structure of the nitrogenase-like dark operative protochlorophyllide oxidoreductase catalytic complex (ChlN/ChlB)2.

During (bacterio)chlorophyll biosynthesis of many photosynthetically active organisms, dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the two-electron reduction of ring D of protochlorophyllide to form chlorophyllide. DPOR is composed of the subunits ChlL, ChlN, and ChlB. Homodimeric ChlL(2) bearing an intersubunit [4Fe-4S] cluster is an ATP-dependent reductase transferring single electrons to the heterotetrameric (ChlN/ChlB)(2) complex. The latter contains two intersubunit [4Fe-4S] clusters and two protochlorophyllide binding sites, respectively. Here we present the crystal structure of the catalytic (ChlN/ChlB)(2) complex of DPOR from the cyanobacterium Thermosynechococcus elongatus at a resolution of 2.4 A. Subunits ChlN and ChlB exhibit a related architecture of three subdomains each built around a central, parallel beta-sheet surrounded by alpha-helices. The (ChlN/ChlB)(2) crystal structure reveals a [4Fe-4S] cluster coordinated by an aspartate oxygen alongside three cysteine ligands. Two equivalent substrate binding sites enriched in aromatic residues for protochlorophyllide substrate binding are located at the interface of each ChlN/ChlB half-tetramer. The complete octameric (ChlN/ChlB)(2)(ChlL(2))(2) complex of DPOR was modeled based on the crystal structure and earlier functional studies. The electron transfer pathway via the various redox centers of DPOR to the substrate is proposed.

Spectrum of MYO7A Mutations in an Indigenous South African Population Further Elucidates the Nonsyndromic Autosomal Recessive Phenotype of DFNB2 to Include Both Homozygous and Compound Heterozygous Mutations.

MYO7A gene encodes unconventional myosin VIIA, which, when mutated, causes a phenotypic spectrum ranging from recessive hearing loss DFNB2 to deaf-blindness, Usher Type 1B (USH1B). MYO7A mutations are reported in nine DFNB2 families to date, none from sub-Saharan Africa.In DNA, from a cohort of 94 individuals representing 92 families from the Limpopo province of South Africa, eight MYO7A variations were detected among 10 individuals. Family studies identified homozygous and compound heterozygous mutations in 17 individuals out of 32 available family members. Four mutations were novel, p.Gly329Asp, p.Arg373His, p.Tyr1780Ser, and p.Pro2126Leufs*5. Two variations, p.Ser617Pro and p.Thr381Met, previously listed as of uncertain significance (ClinVar), were confirmed to be pathogenic. The identified mutations are predicted to interfere with the conformational properties of myosin VIIA through interruption or abrogation of multiple interactions between the mutant and neighbouring residues. Specifically, p.Pro2126Leufs*5, is predicted to abolish the critical site for the interactions between the tail and the motor domain essential for the autoregulation, leaving a non-functional, unregulated protein that causes hearing loss. We have identified MYO7A as a possible key deafness gene among indigenous sub-Saharan Africans. The spectrum of MYO7A mutations in this South African population points to DFNB2 as a specific entity that may occur in a homozygous or in a compound heterozygous state.

Structural basis for autoinhibition and activation of Auto, a virulence-associated peptidoglycan hydrolase of Listeria monocytogenes.

During a bacterial infection, each successive step is orchestrated by a dedicated set of virulence factors. In Gram-positive bacteria, the presentation or release of such factors is crucially dependent on the continual remodelling of the cell wall. We have investigated the autolysin or peptidoglycan hydrolase Auto (Lmo1076) from the human pathogen Listeria monocytogenes to structurally and biochemically underpin its role in host cell invasion. We demonstrate that Auto is an N-acetylglucosaminidase, that it is autoinhibited when newly secreted but activated by proteolytic cleavage, that it has an acidic pH optimum and that it preferentially cleaves acetylated over de-acetylated peptidoglycan. The crystal structure of Auto, the first for glycoside hydrolase family 73, and the first for a listerial autolysin, indicates that autoinhibition is due to an N-terminal alpha-helix unique to Auto that physically blocks the substrate-binding cleft. We identify Glu122 and Glu156 as the two catalytically essential carboxylate groups. The physical properties of Auto as well as its localization to lipoteichoic acid by its four C-terminal GW modules imply cell wall degradation by Auto to be highly co-ordinated. Its spatio-temporally controlled activation and localized activity in an acidified environment indicate that it facilitates remodelling of the cell wall and may be involved in co-ordinating the release of virulence factors at specific stages of an infection.

Structure of the heme biosynthetic Pseudomonas aeruginosa porphobilinogen synthase in complex with the antibiotic alaremycin.

The recently discovered antibacterial compound alaremycin, produced by Streptomyces sp. A012304, structurally closely resembles 5-aminolevulinic acid, the substrate of porphobilinogen synthase. During the initial steps of heme biosynthesis, two molecules of 5-aminolevulinic acid are asymmetrically condensed to porphobilinogen. Alaremycin was found to efficiently inhibit the growth of both Gram-negative and Gram-positive bacteria. Using the newly created heme-permeable strain Escherichia coli CSA1, we are able to uncouple heme biosynthesis from bacterial growth and demonstrate that alaremycin targets the heme biosynthetic pathway. Further studies focused on the activity of alaremycin against the opportunistic pathogenic bacterium Pseudomonas aeruginosa. The MIC of alaremycin was determined to be 12 mM. Alaremycin was identified as a direct inhibitor of recombinant purified P. aeruginosa porphobilinogen synthase and had a K(i) of 1.33 mM. To understand the molecular basis of alaremycin's antibiotic activity at the atomic level, the P. aeruginosa porphobilinogen synthase was cocrystallized with the alaremycin. At 1.75-A resolution, the crystal structure reveals that the antibiotic efficiently blocks the active site of porphobilinogen synthase. The antibiotic binds as a reduced derivative of 5-acetamido-4-oxo-5-hexenoic acid. The corresponding methyl group is, however, not coordinated by any amino acid residues of the active site, excluding its functional relevance for alaremycin inhibition. Alaremycin is covalently bound by the catalytically important active-site lysine residue 260 and is tightly coordinated by several active-site amino acids. Our data provide a solid structural basis to further improve the activity of alaremycin for rational drug design. Potential approaches are discussed.

Glutamate recognition and hydride transfer by Escherichia coli glutamyl-tRNA reductase.

The initial step of tetrapyrrole biosynthesis in Escherichia coli involves the NADPH-dependent reduction by glutamyl-tRNA reductase (GluTR) of tRNA-bound glutamate to glutamate-1-semialdehyde. We evaluated the contribution of the glutamate moiety of glutamyl-tRNA to substrate specificity in vitro using a range of substrates and enzyme variants. Unexpectedly, we found that tRNA(Glu) mischarged with glutamine was a substrate for purified recombinant GluTR. Similarly unexpectedly, the substitution of amino acid residues involved in glutamate side chain binding (S109A, T49V, R52K) or in stabilizing the arginine 52 glutamate interaction (glutamate 54 and histidine 99) did not abrogate enzyme activity. Replacing glutamine 116 and glutamate 114, involved in glutamate-enzyme interaction near the aminoacyl bond to tRNA(Glu), by leucine and lysine, respectively, however, did abolish reductase activity. We thus propose that the ester bond between glutamate and tRNA(Glu) represents the crucial determinant for substrate recognition by GluTR, whereas the necessity for product release by a 'back door' exit allows for a degree of structural variability in the recognition of the amino acid moiety. Analyzing the esterase activity, which occured in the absence of NADPH, of GluTR variants using the substrate 4-nitrophenyl acetate confirmed the crucial role of cysteine 50 for thioester formation. Finally, the GluTR variant Q116L was observed to lack reductase activity whereas esterase activity was retained. Structure-based molecular modeling indicated that glutamine 116 may be crucial in positioning the nicotinamide group of NADPH to allow for productive hydride transfer to the substrate. Our data thus provide new information about the distinct function of active site residues of GluTR from E. coli.

Evolutionary relationship between initial enzymes of tetrapyrrole biosynthesis.

Glutamate-1-semialdehyde 2,1-aminomutase (GSAM) is the second enzyme in the C(5) pathway of tetrapyrrole biosynthesis found in most bacteria, in archaea and in plants. It catalyzes the transamination of glutamate-1-semialdehyde to 5-aminolevulinic acid (ALA) in a pyridoxal 5'-phosphate (PLP)-dependent manner. We present the crystal structure of GSAM from the thermophilic cyanobacterium Thermosynechococcus elongatus (GSAM(Tel)) in its PLP-bound form at 2.85A resolution. GSAM(Tel) is a symmetric homodimer, whereas GSAM from Synechococcus (GSAM(Syn)) has been described as asymmetric. The symmetry of GSAM(Tel) thus challenges the previously proposed negative cooperativity between monomers of this enzyme. Furthermore, GSAM(Tel) reveals an extensive flexible region at the interface of the proposed complex of GSAM with glutamyl-tRNA reductase (GluTR), the preceding enzyme in tetrapyrrole biosynthesis. Compared to GSAM(Syn), the monomers of GSAM(Tel) are rotated away from each other along the dimerization interface by 10 degrees . The associated flexibility of GSAM may be essential for complex formation with GluTR to occur. Unexpectedly, we find that GSAM is structurally related to 5-aminolevulinate synthase (ALAS), the ALA-producing enzyme in the Shemin pathway of alpha-proteobacteria and non-plant eukaryotes. This structural relationship applies also to the corresponding subfamilies of PLP-dependent enzymes. We thus propose that the CoA-subfamily (including ALAS) and the aminotransferase subfamily II (including GSAM) are evolutionarily closely related and that ALAS may thus have evolved from GSAM.

Crystal structure of a non-discriminating glutamyl-tRNA synthetase.

Error-free protein biosynthesis is dependent on the reliable charging of each tRNA with its cognate amino acid. Many bacteria, however, lack a glutaminyl-tRNA synthetase. In these organisms, tRNA(Gln) is initially mischarged with glutamate by a non-discriminating glutamyl-tRNA synthetase (ND-GluRS). This enzyme thus charges both tRNA(Glu) and tRNA(Gln) with glutamate. Discriminating GluRS (D-GluRS), found in some bacteria and all eukaryotes, exclusively generates Glu-tRNA(Glu). Here we present the first crystal structure of a non-discriminating GluRS from Thermosynechococcus elongatus (ND-GluRS(Tel)) in complex with glutamate at a resolution of 2.45 A. Structurally, the enzyme shares the overall architecture of the discriminating GluRS from Thermus thermophilus (D-GluRS(Tth)). We confirm experimentally that GluRS(Tel) is non-discriminating and present kinetic parameters for synthesis of Glu-tRNA(Glu) and of Glu-tRNA(Gln). Anticodons of tRNA(Glu) (34C/UUC36) and tRNA(Gln) (34C/UUG36) differ only in base 36. The pyrimidine base of C36 is specifically recognized in D-GluRS(Tth) by the residue Arg358. In ND-GluRS(Tel) this arginine residue is replaced by glycine (Gly366) presumably allowing both cytosine and the bulkier purine base G36 of tRNA(Gln) to be tolerated. Most other ND-GluRS share this structural feature, leading to relaxed substrate specificity.

Tracking the evolution of porphobilinogen synthase metal dependence in vitro.

Metal ions are indispensable cofactors for chemical catalysis by a plethora of enzymes. Porphobilinogen synthases (PBGSs), which catalyse the second step of tetrapyrrole biosynthesis, are grouped according to their dependence on Zn(2+). Using site-directed mutagenesis, we embarked on transforming Zn(2+)-independent Pseudomonas aeruginosa PBGS into a Zn(2+)-dependent enzyme. Nine PBGS variants were generated by permutationally introducing three cysteine residues and a further two residues into the active site of the enzyme to match the homologous Zn(2+)-containing PBGS from Escherichia coli. Crystal structures of seven enzyme variants were solved to elucidate the nature of Zn(2+) coordination at high resolution. The three single-cysteine variants were invariably found to be enzymatically inactive and only one (D139C) was found to bind detectable amounts of Zn(2+). The double mutant A129C/D139C is enzymatically active and binds Zn(2+) in a tetrahedral coordination. Structurally and functionally it mimics mycobacterial PBGS, which bears an equivalent Zn(2+)-coordination site. The remaining two double mutants, without known natural equivalents, reveal strongly distorted tetrahedral Zn(2+)-binding sites. Variant A129C/D131C possesses weak PBGS activity while D131C/D139C is inactive. The triple mutant A129C/D131C/D139C, finally, displays an almost ideal tetrahedral Zn(2+)-binding geometry and a significant Zn(2+)-dependent enzymatic activity. Two additional amino acid exchanges further optimize the active site architecture towards the E.coli enzyme with an additional increase in activity. Our study delineates the potential evolutionary path between Zn(2+)-free and Zn(2+)-dependent PBGS enyzmes showing that the rigid backbone of PBGS enzymes is an ideal framework to create or eliminate metal dependence through a limited number of amino acid exchanges.

Topological assignment of the N-terminal extension of plasma gelsolin to the gelsolin surface.

The actin-binding protein gelsolin is highly conserved in vertebrates and exists in two isoforms, a cytoplasmic and an extracellular variant, generated by alternative splicing. In mammals, these isoforms differ only by an N-terminal extension in plasma gelsolin, a short sequence of up to 25 amino acids. Cells and tissues may contain both variants, as plasma gelsolin is secreted by many cell types. The tertiary structure of equine plasma gelsolin has been elucidated, but without any information on the N-terminal extension. In this paper, we present topographical data on the N-terminal extension, derived using a biochemical and immunological approach. For this purpose, a monoclonal antibody was generated that exclusively recognizes cytoplasmic gelsolin but not the extracellular variant and thus allows isoform-specific immunodetection and quantification of cytoplasmic gelsolin in the presence of plasma gelsolin. Using limited proteolysis and pepscan analysis, we mapped the binding epitope and localized it within two regions in segment 1 of the cytoplasmic gelsolin sequence: Tyr34-Ile45 and Leu64-Ile78. In the tertiary structure of the cytoplasmic variant, these sequences are mutually adjacent and located in the proximity of the N-terminus. We therefore conclude that the binding site of the antibody is covered by the N-terminal extension in plasma gelsolin and thus sterically hinders antibody binding. Our results allow for a topological model of the N-terminal extension on the surface of the gelsolin molecule, which was unknown previously.

Expression and production of soluble Mycobacterium tuberculosis H37Rv mycosin-3.

Mycobacteria encode five type VII secretion system (T7SS) or ESX for nutrient acquisition and virulence. Mycosins are membrane-anchored components of ESX with serine protease activity but an unidentified substrate range. Establishing the substrate specificity of individual mycosins will help to elucidate individual ESX functions. Mycosin-1 and -3 orthologues from two environmental mycobacterial species, Mycobacterium smegmatis and Mycobacterium thermoresistibile, have been heterologously produced, but mycosins from Mycobacterium tuberculosis (Mtb) remain to be studied. Here we describe the successful production of Mtb mycosin-3 as a first step in investigating its structure and function.

Structure and function of radical SAM enzymes.

'Radical SAM' enzymes juxtapose a [4Fe-4S] cluster and S-adenosyl-l-methionine (SAM) to generate catalytic 5'-deoxyadenosyl radicals. The crystal structures of oxygen-independent coproporphyrinogen III oxidase HemN and biotin synthase reveal the positioning of both cofactors with respect to each other and relative to the surrounding protein environment. Each is found in an unprecedented coordination environment including the direct ligation of the [4Fe-4S] cluster by the amino nitrogen and one carboxylate oxygen of the methionine moiety of SAM, as observed for other members of the Radical SAM family by ENDOR. The availability of two protein structures supported by biochemical and biophysical data underscores common features, anticipating the structural elements of other family members. Remaining differences emphasize the plasticity of the protein scaffold in functionally accommodating 600 family members.