Lyase Domain Research Articles

Site-Directed Mutagenesis-Based Functional Analysis and Characterization of Endolytic Lyase Activity of N- and C-Terminal Domains of a Novel Oligoalginate Lyase from Sphingomonas sp. MJ-3 Possessing Exolytic Lyase Activity in the Intact Enzyme.

A novel oligoalginate lyase from a marine bacterium, Sphingomonas sp. strain MJ-3, exhibited a unique alginate degradation activity that completely depolymerizes alginate to monomers through the formation of oligomers. In order to reveal the reason why MJ-3 oligoalginate can exhibit both endolytic and exolytic alginate lyase activities, ten mutants were developed and characterized on the basis of homology modeling. When the recombinant cell lysates containing the mutated proteins of MJ-3 oligoalginate lyase were allowed to react with alginate, the Asn177Ala, His178Ala, Tyr234Phe, His389Ala, and Tyr426Phe mutants showed reduced oligoalginate lyase activity, whereas the Arg236Ala mutant exhibited endolytic activity. Interestingly, the overexpressed Arg236Ala protein (79.6 kDa) was proteolytically cleaved into two fragments, i.e., the N-terminal 32.0-kDa and the C-terminal 47.6-kDa fragments. Both the purified N-terminal and C-terminal fragments showed endolytic lyase activity. They preferentially degraded a heteropolymeric (polyMG) block than poly-β-D-mannuronate (polyM) or poly-α-L-guluronate (polyG) blocks. These results suggest that the oligoalginate lyase activity of MJ-3 enzyme is derived from the cooperative interaction between the N- and C-terminal endolytic alginate lyase domains in the intact enzyme.

Mercury Reduction and Methyl Mercury Degradation by the Soil Bacterium Xanthobacter autotrophicus Py2.

Two previously uncharacterized potential broad-spectrum mercury (Hg) resistance operons (mer) are present on the chromosome of the soil Alphaproteobacteria Xanthobacter autotrophicus Py2. These operons, mer1 and mer2, contain two features which are commonly found in mer operons in the genomes of soil and marine Alphaproteobacteria, but are not present in previously characterized mer operons: a gene for the mercuric reductase (MerA) that encodes an alkylmercury lyase domain typical of those found on the MerB protein, and the presence of an additional gene, which we are calling merK, with homology to glutathione reductase. Here, we demonstrate that Py2 is resistant to 0.2 μM inorganic mercury [Hg(II)] and 0.05 μM methylmercury (MeHg). Py2 is capable of converting MeHg and Hg(II) to elemental mercury [Hg(0)], and reduction of Hg(II) is induced by incubation in sub toxic concentrations of Hg(II). Transcription of the merA genes increased with Hg(II) treatment, and in both operons merK resides on the same polycistronic mRNA as merA. We propose the use of Py2 as a model system for studying the contribution of mer to Hg mobility in soil and marine ecosystems.

C-S bond cleavage by a polyketide synthase domain

Leinamycin (LNM) is a sulfur-containing antitumor antibiotic featuring an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a thiazole-containing 18-membered lactam ring. The 1,3-dioxo-1,2-dithiolane moiety is essential for LNM's antitumor activity, by virtue of its ability to generate an episulfonium ion intermediate capable of alkylating DNA. We have previously cloned and sequenced the lnm gene cluster from Streptomyces atroolivaceus S-140. In vivo and in vitro characterizations of the LNM biosynthetic machinery have since established that: (i) the 18-membered macrolactam backbone is synthesized by LnmP, LnmQ, LnmJ, LnmI, and LnmG, (ii) the alkyl branch at C-3 of LNM is installed by LnmK, LnmL, LnmM, and LnmF, and (iii) leinamycin E1 (LNM E1), bearing a thiol moiety at C-3, is the nascent product of the LNM hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS). Sulfur incorporation at C-3 of LNM E1, however, has not been addressed. Here we report that: (i) the bioinformatics analysis reveals a pyridoxal phosphate (PLP)-dependent domain, we termed cysteine lyase (SH) domain (LnmJ-SH), within PKS module-8 of LnmJ; (ii) the LnmJ-SH domain catalyzes C-S bond cleavage by using l-cysteine and l-cysteine S-modified analogs as substrates through a PLP-dependent β-elimination reaction, establishing l-cysteine as the origin of sulfur at C-3 of LNM; and (iii) the LnmJ-SH domain, sharing no sequence homology with any other enzymes catalyzing C-S bond cleavage, represents a new family of PKS domains that expands the chemistry and enzymology of PKSs and might be exploited to incorporate sulfur into polyketide natural products by PKS engineering.

Key steps in type III secretion system (T3SS) towards translocon assembly with potential sensor at plant plasma membrane.

Many plant- and animal-pathogenic Gram-negative bacteria employ the type III secretion system (T3SS) to translocate effector proteins from bacterial cells into the cytosol of eukaryotic host cells. The effector translocation occurs through an integral component of T3SS, the channel-like translocon, assembled by hydrophilic and hydrophobic proteinaceous translocators in a two-step process. In the first, hydrophilic translocators localize to the tip of a proteinaceous needle in animal pathogens, or a proteinaceous pilus in plant pathogens, and associate with hydrophobic translocators, which insert into host plasma membranes in the second step. However, the pilus needs to penetrate plant cell walls in advance. All hydrophilic translocators so far identified in plant pathogens are characteristic of harpins: T3SS accessory proteins containing a unitary hydrophilic domain or an additional enzymatic domain. Two-domain harpins carrying a pectate lyase domain potentially target plant cell walls and facilitate the penetration of the pectin-rich middle lamella by the bacterial pilus. One-domain harpins target plant plasma membranes and may play a crucial role in translocon assembly, which may also involve contrapuntal associations of hydrophobic translocators. In all cases, sensory components in the target plasma membrane are indispensable for the membrane recognition of translocators and the functionality of the translocon. The conjectural sensors point to membrane lipids and proteins, and a phosphatidic acid and an aquaporin are able to interact with selected harpin-type translocators. Interactions between translocators and their sensors at the target plasma membrane are assumed to be critical for translocon assembly.

N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis.

The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5'-2-deoxyribose-5-phosphate lyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or an 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated -1 or -2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of -2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of -1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase within the non-homologous end joining pathway.

Genome-wide search for eliminylating domains reveals novel function for BLES03-like proteins.

Bacterial phosphothreonine lyases catalyze a novel posttranslational modification involving formation of dehydrobutyrine/dehyroalanine by β elimination of the phosphate group of phosphothreonine or phosphoserine residues in their substrate proteins. Though there is experimental evidence for presence of dehydro amino acids in human proteins, no eukaryotic homologs of these lyases have been identified as of today. A comprehensive genome-wide search for identifying phosphothreonine lyase homologs in eukaryotes was carried out. Our fold-based search revealed structural and catalytic site similarity between bacterial phosphothreonine lyases and BLES03 (basophilic leukemia-expressed protein 03), a human protein with unknown function. Ligand induced conformational changes similar to bacterial phosphothreonine lyases, and movement of crucial arginines in the loop region to the catalytic pocket upon binding of phosphothreonine-containing peptides was seen during docking and molecular dynamics studies. Genome-wide search for BLES03 homologs using sensitive profile-based methods revealed their presence not only in eukaryotic classes such as chordata and fungi but also in bacterial and archaebacterial classes. The synteny of these archaebacterial BLES03-like proteins was remarkably similar to that of type IV lantibiotic synthetases which harbor LanL-like phosphothreonine lyase domains. Hence, context-based analysis reinforced our earlier sequence/structure-based prediction of phosphothreonine lyase catalytic function for BLES03. Our in silico analysis has revealed that BLES03-like proteins with previously unknown function are novel eukaryotic phosphothreonine lyases involved in biosynthesis of dehydro amino acids, whereas their bacterial and archaebacterial counterparts might be involved in biosynthesis of natural products similar to lantibiotics.

Genetic determinants of amidating enzyme activity and its relationship with metal cofactors in human serum.

Backgroundα-amidation is a final, essential step in the biosynthesis of about half of all peptide hormones and neurotransmitters. Peptidylglycine α-amidating monooxygenase (PAM), with enzymatic domains that utilize Cu and Zn, is the only enzyme that catalyzes this reaction. PAM activity is detected in serum, but its significance and utility as a clinical biomarker remain unexplored.MethodsWe used well-established enzymatic assays specific for the peptidylglycine-α -hydroxylating monooxygenase (PHM) and peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domains of PAM to quantify amidating activity in the sera of 144 elderly men. Relationships between PHM and PAL activity and serum levels of their respective active-site metals, Cu and Zn, were analyzed. Study participants were also genotyped for eight non-coding single nucleotide polymorphisms (SNPs) in PAM, and relationships between genotype and serum enzyme activity and metal levels were analyzed.ResultsSerum PHM and PAL activities were normally distributed and correlated linearly with each other. Serum PAL activity, but not serum PHM activity, correlated with serum Cu; neither activity correlated with serum Zn. Study subjects possessing the minor alleles for rs32680 had lower PHM and PAL activities, and subjects with minor alleles for rs11952361 and rs10515341 had lower PHM activities.ConclusionsOur results characterize large variation in serum amidating activity and provide unique insight into its potential origin and determinants. Common non-coding polymorphisms affect serum amidating activity and Cu levels. Serum amidating activity should be explored as a biomarker for functionality in the elderly and in additional study groups.

Structure and Mechanism of DNA Polymerase β

DNA polymerase (pol) β is a small eukaryotic DNA polymerase composed of two domains. Each domain contributes an enzymatic activity (DNA synthesis and deoxyribose phosphate lyase) during the repair of simple base lesions. These domains are termed the polymerase and lyase domains, respectively. Pol β has been an excellent model enzyme for studying the nucleotidyl transferase reaction and substrate discrimination at a molecular level. In this review, recent crystallographic studies of pol β in various liganded and conformational states during the insertion of right and wrong nucleotides as well as during the bypass of damaged DNA (apurinic sites and 8-oxoguanine) are described. Structures of these catalytic intermediates provide unexpected insights into mechanisms by which DNA polymerases enhance genome stability. These structures also provide an improved framework that permits computational studies to facilitate the interpretation of detailed kinetic analyses of this model enzyme.

Substrate Rescue of DNA Polymerase β Containing a Catastrophic L22P Mutation

DNA polymerase (pol) β is a multidomain enzyme with two enzymatic activities that plays a central role in the overlapping base excision repair and single-strand break repair pathways. The high frequency of pol β variants identified in tumor-derived tissues suggests a possible role in the progression of cancer, making the determination of the functional consequences of these variants of interest. Pol β containing a proline substitution for leucine 22 in the lyase domain (LD), identified in gastric tumors, has been reported to exhibit severe impairment of both lyase and polymerase activities. Nuclear magnetic resonance (NMR) spectroscopic evaluations of both pol β and the isolated LD containing the L22P mutation demonstrate destabilization sufficient to result in LD-selective unfolding with minimal structural perturbations to the polymerase domain. Unexpectedly, addition of single-stranded or hairpin DNA resulted in partial refolding of the mutated lyase domain, both in isolation and for the full-length enzyme. Further, formation of an abortive ternary complex using Ca2+ and a complementary dNTP indicates that the fraction of pol β(L22P) containing the folded LD undergoes conformational activation similar to that of the wild-type enzyme. Kinetic characterization of the polymerase activity of L22P pol β indicates that the L22P mutation compromises DNA binding, but nearly wild-type catalytic rates can be observed at elevated substrate concentrations. The organic osmolyte trimethylamine N-oxide (TMAO) is similarly able to induce folding and kinetic activation of both polymerase and lyase activities of the mutant. Kinetic data indicate synergy between the TMAO cosolvent and substrate binding. NMR data indicate that the effect of the DNA results primarily from interaction with the folded LD(L22P), while the effect of the TMAO results primarily from destabilization of the unfolded LD(L22P). These studies illustrate that substrate-induced catalytic activation of pol β provides an optimal enzyme conformation even in the presence of a strongly destabilizing point mutation. Accordingly, it remains to be determined whether this mutation alters the threshold of cellular repair activity needed for routine genome maintenance or whether the “inactive” variant interferes with DNA repair.

The HrpW protein of Lonsdalea quercina N‐5‐1 has pectate lyase activity and is required for full bacterial virulence

Lonsdalea quercina N-5-1 is a bacterial pathogen that causes poplar bark cankers. It has been isolated from the branch of Populus × euramericana cv. "74/76" in Henan, China. Previous studies have revealed that the Type III secretion system (T3SS) acts as an essential pathogenic factor in L. quercina N-5-1. HrpW is a putative effector of T3SS in strain N-5-1, which has a typical harpin domain at the amino terminal and a pectate lyase (Pel) domain at its carboxyl terminal. Genetic evidence had shown that, compared to the wild-type and the complementary strain, the hrpW mutation causes a small but significant reduction in virulence when inoculated on the poplar branches. The amino terminal domain of HrpW was found to trigger tobacco hypersensitive response, but the carboxyl terminal domain of HrpW was not. Unlike most HrpW homologs in other bacteria, the carboxyl terminal domain of HrpW of strain N-5-1 exhibited detectable pectate lyase activity. Site-direction mutations (W104A, W171M) further demonstrated that two tryptophan residues were essential to its pectate lyase activity. The results of the present work suggest that HrpW in L. quercina N-5-1 possesses pectate lyase activity and acts as a nonessential but important pathogenic factor in poplar bark canker disease.

The strawberry (Fragaria×ananassa) fruit-specific rhamnogalacturonate lyase 1 (FaRGLyase1) gene encodes an enzyme involved in the degradation of cell-wall middle lamellae

Pectins are essential components of primary plant cell walls and middle lamellae, and are related to the consistency of the fruit and its textural changes during ripening. In fact, strawberries become soft as the middle lamellae of cortical parenchyma cells are extensively degraded during ripening, leading to the observed short post-harvest shelf life. Using a custom-made oligonucleotide-based strawberry microarray platform, a putative rhamnogalacturonate lyase gene (FaRGlyase1) was identified. Bioinformatic analysis of the FaRGlyase1 sequence allowed the identification of a conserved rhamnogalacturonate lyase domain, which was also present in other putative RGlyase sequences deposited in the databases. Expression of FaRGlyase1 occurred mainly in the receptacle, concurrently with ripening, and it was positively regulated by abscisic acid and negatively by auxins. FaRGLyase1 gene expression was transiently silenced by injecting live Agrobacterium cells harbouring RNA interference constructs into fruit receptacles. Light and electron microscopy analyses of these transiently silenced fruits revealed that this gene is involved in the degradation of pectins present in the middle lamella region between parenchymatic cells. In addition, genetic linkage association analyses in a strawberry-segregating population showed that FaRGLyase1 is linked to a quantitative trait loci linkage group related to fruit hardness and firmness. The results showed that FaRGlyase1 could play an important role in the fruit ripening-related softening process that reduces strawberry firmness and post-harvest life.

The Acinetobacter baumannii entA Gene Located Outside the Acinetobactin Cluster Is Critical for Siderophore Production, Iron Acquisition and Virulence

Acinetobacter baumannii causes severe infections in compromised patients, who present an iron-limited environment that controls bacterial growth. This pathogen has responded to this restriction by expressing high-affinity iron acquisition systems including that mediated by the siderophore acinetobactin. Gene cloning, functional assays and biochemical tests showed that the A. baumannii genome contains a single functional copy of an entA ortholog. This gene, which is essential for the biosynthesis of the acinetobactin precursor 2,3-dihydroxybenzoic acid (DHBA), locates outside of the acinetobactin gene cluster, which otherwise harbors all genes needed for acinetobactin biosynthesis, export and transport. In silico analyses and genetic complementation tests showed that entA locates next to an entB ortholog, which codes for a putative protein that contains the isochorismatase lyase domain, which is needed for DHBA biosynthesis from isochorismic acid, but lacks the aryl carrier protein domain, which is needed for tethering activated DHBA and completion of siderophore biosynthesis. Thus, basF, which locates within the acinetobactin gene cluster, is the only fully functional entB ortholog present in ATCC 19606T. The differences in amino acid length and sequences between these two EntB orthologs and the differences in the genetic context within which the entA and entB genes are found in different A. baumannii isolates indicate that they were acquired from different sources by horizontal transfer. Interestingly, the AYE strain proved to be a natural entA mutant capable of acquiring iron via an uncharacterized siderophore-mediated system, an observation that underlines the ability of different A. baumannii isolates to acquire iron using different systems. Finally, experimental infections using in vivo and ex vivo models demonstrate the role of DHBA and acinetobactin intermediates in the virulence of the ATCC 19606T cells, although to a lesser extent when compared to the responses obtained with bacteria producing and using fully matured acinetobactin to acquire iron.

Substrate-Dependent Millisecond Domain Motions in DNA Polymerase β

DNA polymerase β (Pol β) is a 39-kDa enzyme that performs the vital cellular function of repairing damaged DNA. Mutations in Pol β have been linked to various cancers, and these mutations are further correlated with altered Pol β enzymatic activity. The fidelity of correct nucleotide incorporation into damaged DNA is essential for Pol β repair function, and several studies have implicated conformational changes in Pol β as a determinant of this repair fidelity. In this work, the rate constants for domain motions in Pol β have been determined by solution NMR relaxation dispersion for the apo and substrate-bound, binary forms of Pol β. In apo Pol β, molecular motions, primarily isolated to the DNA lyase domain, are observed to occur at 1400 s−1. Additional analysis suggests that these motions allow apo Pol β to sample a conformation similar to the gapped DNA-substrate-bound form. Upon binding DNA, these lyase domain motions are significantly quenched, whereas evidence for conformational motions in the polymerase domain becomes apparent. These NMR studies suggest an alteration in the dynamic landscape of Pol β due to substrate binding. Moreover, a number of the flexible residues identified in this work are also the location of residues, which upon mutation lead to cancer phenotypes in vivo, which may be due to the intimate role of protein motions in Pol β fidelity.

Probing the Production of Amidated Peptides following Genetic and Dietary Copper Manipulations

Amidated neuropeptides play essential roles throughout the nervous and endocrine systems. Mice lacking peptidylglycine α-amidating monooxygenase (PAM), the only enzyme capable of producing amidated peptides, are not viable. In the amidation reaction, the reactant (glycine-extended peptide) is converted into a reaction intermediate (hydroxyglycine-extended peptide) by the copper-dependent peptidylglycine-α-hydroxylating monooxygenase (PHM) domain of PAM. The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domain of PAM. PHM and PAL are stitched together in vertebrates, but separated in some invertebrates such as Drosophila and Hydra. In addition to its luminal catalytic domains, PAM includes a cytosolic domain that can enter the nucleus following release from the membrane by γ-secretase. In this work, several glycine- and hydroxyglycine-extended peptides as well as amidated peptides were qualitatively and quantitatively assessed from pituitaries of wild-type mice and mice with a single copy of the Pam gene (PAM+/−) via liquid chromatography-mass spectrometry-based methods. We provide the first evidence for the presence of a peptidyl-α-hydroxyglycine in vivo, indicating that the reaction intermediate becomes free and is not handed directly from PHM to PAL in vertebrates. Wild-type mice fed a copper deficient diet and PAM+/− mice exhibit similar behavioral deficits. While glycine-extended reaction intermediates accumulated in the PAM+/− mice and reflected dietary copper availability, amidated products were far more prevalent under the conditions examined, suggesting that the behavioral deficits observed do not simply reflect a lack of amidated peptides.

Genetic and biochemical analyses of chromosome and plasmid gene homologues encoding ICL and ArCP domains in Vibrio anguillarum strain 775

Anguibactin, the siderophore produced by Vibrioanguillarum 775 is synthesized from 2,3-dihydroxybenzoic acid (DHBA), cysteine and hydroxyhistamine via a nonribosomal peptide synthetase (NRPS) mechanism. Most of the genes encoding anguibactin biosynthetic proteins are harbored by the pJM1 plasmid. In this work we report the identification of a homologue of the plasmid-encoded angB on the chromosome of strain 775. The product of both genes harbor an isochorismate lyase (ICL) domain that converts isochorismic acid to 2,3-dihydro-2,3-dihydroxybenzoic acid, one of the steps of DHBA synthesis. We show in this work that both ICL domains are functional in the production of DHBA in V. anguillarum as well as in E. coli. Substitution by alanine of the aspartic acid residue in the active site of both ICL domains completely abolishes their isochorismate lyase activity in vivo. The two proteins also carry an aryl carrier protein (ArCP) domain. In contrast with the ICL domains only the plasmid encoded ArCP can participate in anguibactin production as determined by complementation analyses and site-directed mutagenesis in the active site of the plasmid encoded protein, S248A. The site-directed mutants, D37A in the ICL domain and S248A in the ArCP domain of the plasmid encoded AngB were also tested in vitro and clearly show the importance of each residue for the domain function and that each domain operates independently.

Mechanistic Studies of Ser/Thr Dehydration Catalyzed by a Member of the LanL Lanthionine Synthetase Family

Members of the LanL family of lanthionine synthetases consist of three catalytic domains, an N-terminal pSer/pThr lyase domain, a central Ser/Thr kinase domain, and a C-terminal lanthionine cyclase domain. The N-terminal lyase domain has sequence homology with members of the OspF family of effector proteins. In this study, the residues in the lyase domain of VenL that are conserved in the active site of OspF proteins were mutated to evaluate their importance for catalysis. In addition, residues that are fully conserved in the LanL family but not in the OspF family were mutated. Activity assays with these mutant proteins are consistent with a model in which Lys80 in VenL deprotonates the α-proton of pSer/pThr residues to initiate the elimination reaction. Lys51 is proposed to activate this proton by coordination to the carbonyl of the pSer/pThr, and His53 is believed to protonate the phosphate leaving group. These functions are very similar to the corresponding homologous residues in OspF proteins. On the other hand, recognition of the phosphate group of pSer/pThr appears to be achieved differently in VenL than in the OspF proteins. Arg156 and Lys103 are thought to interact with the phosphate group on the basis of a structural homology model.

NMR study of the effect of Zn on conformational activation of rat DNA polymerase β

The DNA repair enzyme DNA Polymerase β (Polβ) fills gapped DNA using a two‐metal ion (usually Mg2+) mechanism. Crystal structures show that the transition from an “open” binary complex (Polβ·gapped DNA) to a “closed” ternary complex (Polβ·gapped DNA·dNTP) activates formation of the active site geometry involved in Mg2+ coordination. Other divalent metals can also function in place of magnesium, and some can cause a decrease in fidelity. We have investigated the effect of Zn by using [methyl‐13C] methionine‐labeled Polβ in NMR experiments to study the conformational changes undergone by Polβ in solution during its catalytic cycle The methionine probes are located in the lyase domain (M18), and in different parts of the polymerase domain, i.e. the catalytic subdomain (M155, M158, M191, and M236); and the nascent base‐pair binding subdomain (M282). The transitions that correspond to the “open” to “closed” structures (with Mg2+ present) can be monitored by the chemical shifts of the methionine methyl groups. In particular, the large chemical shift change and broadening of the M282 resonance detects the rearrangement of helix N (in the nascent base‐pair binding subdomain) induced by the formation of the activated ternary complex. In this work we show that these same responses can be achieved for a binary complex in the presence of Zn2+ ions. (Supported by the Intramural Research Program of the National Institutes of Health)

PopW of Ralstonia solanacearum, a new two‐domain harpin targeting the plant cell wall

Harpins are extracellular glycine-rich proteins eliciting a hypersensitive response (HR). In this study, we identified a new harpin, PopW, from Ralstonia solanacearum strain ZJ3721. This 380-amino-acid protein is acidic, rich in glycine and serine, and lacks cysteine. When infiltrated into the leaves of tobacco (non-host), PopW induced a rapid tissue collapse via a heat-stable but protease-sensitive HR-eliciting activity. PopW has an N-terminal harpin domain (residues 1-159) and a C-terminal pectate lyase (PL) domain (residues 160-366); its HR-eliciting activity depends on its N-terminal domain. Analyses of subcellular localization and plasmolysis demonstrated that PopW targeted the onion cell wall. This was further confirmed by its ability to specifically bind to calcium pectate, a major component of the plant cell wall. However, PopW had no detectable PL activity. Western blotting revealed that PopW was secreted by the type III secretion system in an hrpB-dependent manner. Gene sequencing indicated that popW is conserved among 20 diverse strains of R. solanacearum. A popW-deficient mutant retained the ability of wild-type strain ZJ3721 to elicit HR in tobacco and to cause wilt disease in tomato (a host). We conclude that PopW is a new cell wall-associated, hrpB-dependent, two-domain harpin that is conserved across the R. solanacearum species complex.

Discovery of unique lanthionine synthetases reveals new mechanistic and evolutionary insights.

Author SummaryMany bacteria generate cyclic peptides, which have improved biological activities compared to linear peptides, including higher stability. Lanthionine-containing peptides are one such group, and different members of this group have antibiotic, anti-inflammatory, and anti-viral activities. For example, one lanthionine-containing peptide called nisin has been used to protect food items from harmful bacteria. Two different pathways for the biosynthesis of lanthionine-containing peptides have been described previously. By comparing the DNA sequences of bacterial genomes we reveal a third biosynthetic route that provides further insight into how the biosynthetic pathways for these cyclic peptides have evolved. We characterized the novel lanthionine synthetase utilized in this third pathway in the soil bacterium Streptomyces venezuelae and show that the purified enzyme catalyzes the chemical reactions necessary to turn a linear peptide into a peptide with multiple rings. The discovery of this third biosynthetic pathway widens the scope for the engineering of new lanthionine-containing peptides for potential use in human therapeutics.

Modulation of the Structure, Catalytic Activity, and Fidelity of African Swine Fever Virus DNA Polymerase X by a Reversible Disulfide Switch

African swine fever virus polymerase X (pol X) is the smallest DNA polymerase known (174 amino acids), and its tertiary structure resembles the C-terminal half of prototypical X-family pol beta, which includes a catalytic dNTP-binding site (palm domain) and a finger domain. This structural similarity and the presence of viral genes coding for other base excision repair proteins suggest that pol X functions in a manner similar to pol beta, but inconsistencies concerning pol X catalysis have been reported. We examined the structural and functional properties of two forms of pol X using spectroscopic and kinetic analysis. Using (1)H-(15)N correlated NMR, we unambiguously demonstrated the slow interconversion of pol X between a reduced (pol X(red)) and an oxidized form (pol X(ox)), confirmed by mass spectrometry. Steady-state kinetic analysis revealed that pol X(ox), with a disulfide bond between Cys-81 and Cys-86, has approximately 10-fold lower fidelity than pol X(red) during dNTP insertion opposite a template G. The disulfide linkage is located between two beta-strands in the palm domain, near the putative dNTP-binding site. Structural alignment of pol X with a pol beta ternary structure suggests that the disulfide switch may modulate fidelity by altering the ability of the palm domain to align and stabilize the primer terminus and catalytic metal ion for deprotonation of the 3'-OH group and subsequent phosphoryl transfer. Thus, DNA polymerase fidelity is altered by the redox state of the enzyme and its related conformational changes.