Impact of C-terminal domains of paralogous single-stranded DNA binding proteins from Streptomyces coelicolor on their biophysical properties and biological functions.

Single-stranded DNA-binding proteins (SSB) are crucial in DNA metabolism. While Escherichia coli SSB is extensively studied, the significance of its C-terminal domain has only recently emerged. This study explored the significance of C-domains of two paralogous Ssb proteins in S. coelicolor. Mutational analyses of C-domains uncovered a novel role of SsbA during sporulation-specific cell division and demonstrated that the C-tip is non-essential for survival. In vitro methods revealed altered biophysical and biochemical properties of Ssb proteins with modified C-domains. Determined hydrodynamic properties suggested that the C-domains of SsbA and SsbB occupy a globular position proposed to mediate cooperative binding. Only SsbA was found to form biomolecular condensates independent of the C-tip. Interestingly, the truncated C-domain of SsbA increased the molar enthalpy of unfolding. Additionally, calorimetric titrations revealed that C-domain mutations affected ssDNA binding. Moreover, this analysis showed that the SsbA C-tip aids binding most likely by regulating the position of the flexible C-domain. It also highlighted ssDNA-induced conformational mobility restrictions of all Ssb variants. Finally, the gel mobility shift assay confirmed that the intrinsically disordered linker is essential for cooperative binding of SsbA. These findings highlight the important role of the C-domain in the functioning of SsbA and SsbB proteins.

Evaluation of the Structure-Function Relationship of SGNH Lipase from Streptomyces rimosus by Site-Directed Mutagenesis and Computational Approach.

Streptomyces rimosus extracellular lipase (SrL) is a multifunctional hydrolase belonging to the SGNH family. Here site-directed mutagenesis (SDM) was used for the first time to investigate the functional significance of the conserved amino acid residues Ser10, Gly54, Asn82, Asn213, and His216 in the active site of SrL. The hydrolytic activity of SrL variants was determined using para-nitrophenyl (pNP) esters with C4, C8, and C16 fatty acid chains. Mutation of Ser10, Asn82, or His216, but not Gly54, to Ala abolished lipase activity for all substrates. In contrast, the Asn213Ala variant showed increased enzymatic activity for C8 and C16 pNP esters. Molecular dynamics (MD) simulations showed that the interactions between the long alkyl chain substrate (C16) and Ser10 and Asn82 were strongest in Asn213Ala SrL. In addition to Asn82, Gly54, and Ser10, several new constituents of the substrate binding site were recognized (Lys28, Ser53, Thr89, and Glu212), as well as strong electrostatic interactions between Lys28 and Glu212. In addition to the H bonds Ser10-His216 and His216-Ser214, Tyr11 interacted strongly with Ser10 and His216 in all complexes with an active enzyme form. A previously unknown strong H bond between the catalytically important Asn82 and Gly54 was uncovered, which stabilizes the substrate in an orientation suitable for the enzyme reaction.

Malassezia pachydermatis from brown bear: A comprehensive analysis reveals novel genotypes and distribution of all detected variants in domestic and wild animals.

Malassezia pachydermatis (phylum Basidiomycota, class Malasseziomycetes) is a zoophilic opportunistic pathogen with recognized potential for invasive infections in humans. Although this pathogenic yeast is widespread in nature, it has been primarily studied in domestic animals, so available data on its genotypes in the wild are limited. In this study, 80 yeast isolates recovered from 42 brown bears (Ursus arctos) were identified as M. pachydermatis by a culture-based approach. MALDI-TOF mass spectrometry (MS) was used to endorse conventional identification. The majority of samples exhibited a high score fluctuation, with 42.5% of isolates generating the best scores in the range confident only for genus identification. However, the use of young biomass significantly improved the identification of M. pachydermatis at the species confidence level (98.8%). Importantly, the same MALDI-TOF MS efficiency would be achieved regardless of colony age if the cut-off value was lowered to ≥1.7. Genotyping of LSU, ITS1, CHS2, and ß-tubulin markers identified four distinct genotypes in M. pachydermatis isolates. The most prevalent among them was the genotype previously found in dogs, indicating its transmission potential and adaptation to distantly related hosts. The other three genotypes are described for the first time in this study. However, only one of the genotypes consisted of all four loci with bear-specific sequences, indicating the formation of a strain specifically adapted to brown bears. Finally, we evaluated the specificity of the spectral profiles of the detected genotypes. MALDI-TOF MS exhibited great potential to detect subtle differences between all M. pachydermatis isolates and revealed distinct spectral profiles of bear-specific genotypes.

Phosphoproteome Dynamics of Streptomyces rimosus during Submerged Growth and Antibiotic Production.

Streptomyces rimosus is an industrial streptomycete, best known as a producer of oxytetracycline, one of the most widely used antibiotics. Despite the significant contribution of Streptomyces species to the pharmaceutical industry, most omics analyses have only been conducted on the model organism Streptomyces coelicolor. In recent years, protein phosphorylation on serine, threonine, and tyrosine (Ser, Thr, and Tyr, respectively) has been shown to play a crucial role in the regulation of numerous cellular processes, including metabolic changes leading to antibiotic production and morphological changes. In this study, we performed a comprehensive quantitative (phospho)proteomic analysis during the growth of S. rimosus under conditions of oxytetracycline production and pellet fragmentation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis combined with phosphopeptide enrichment detected a total of 3,725 proteins, corresponding to 45.6% of the proteome and 417 phosphorylation sites from 230 phosphoproteins. Significant changes in abundance during three distinct growth phases were determined for 494 proteins and 98 phosphorylation sites. Functional analysis revealed changes in phosphorylation events of proteins involved in important cellular processes, including regulatory mechanisms, primary and secondary metabolism, cell division, and stress response. About 80% of the phosphoproteins detected during submerged growth of S. rimosus have not yet been reported in streptomycetes, and 55 phosphoproteins were not reported in any prokaryote studied so far. This enabled the creation of a unique resource that provides novel insights into the dynamics of (phospho)proteins and reveals many potential regulatory events during antibiotic production in liquid culture of an industrially important bacterium. IMPORTANCE Streptomyces rimosus is best known as a primary source of oxytetracycline (OTC). The significant global market value of OTC highlights the need for a better understanding of the regulatory mechanisms that lead to production of this antibiotic. Our study provides, for the first time, a detailed insight into the dynamics of (phospho)proteomic profiles during growth and antibiotic production in liquid culture of S. rimosus. Significant changes in protein synthesis and phosphorylation have been revealed for a number of important cellular proteins during the growth stages that coincide with OTC production and morphological changes of this industrially important bacterium. Most of these proteins have not been detected in previous studies. Therefore, our results significantly expand the insight into phosphorylation events associated with important cellular processes and antibiotic production; they also greatly increase the phosphoproteome of streptomycetes and contribute with newly discovered phosphoproteins to the database of prokaryotic phosphoproteomes. This can consequently lead to the design of novel research directions in elucidation of the complex regulatory network in Streptomyces.

Novel arsenic hyper-resistant bacteria from an extreme environment, Crven Dol mine, Allchar, North Macedonia.

Novel hyper-resistant bacteria were isolated from the Crven Dol mine (Allchar, North Macedonia), arsenic-rich extreme environment. Bacteria were recovered from a secondary mineral mixture, an alteration of hydrothermal realgar rich in arsenates (pharmacolite, hornesite, and talmessite). The sample was recovered from the dark part of the mine at 28 m depth. Three bacterial strains and a bacterial consortium were isolated for their capacity to survive exposure to 32 g/L (209 mM) of arsenite, and 176 g/L (564 mM) of arsenate. The 16S rRNA gene analysis identified bacterial isolates as Stenotrophomonas sp. and two Microbacterium spp. This analysis also revealed that bacterial consortium comprise two Bacteriodetes exhibiting similarity to Olivibacter ginsengisoli and to uncultured bacterium, and one γ-proteobacteria with similarity to Luteimonas sp. Among all isolates Stenotrophomonas sp. exhibited the highest tolerance to As compound as well as the capacity to accumulate As inside the cells. Analysis of genes involved in As-resistance showed that recovered isolates possess the genes encoding the ArsB, Acr3(1) and Acr3(2) proteins, indicating that at least a part of their resistance could be ascribed to As-efflux systems described in isolates obtained from human-polluted environments.

Leitmotif: protein motif scanning 2.0.

MOTIVATION: Motif-HMM (mHMM) scanning has been shown to possess unique advantages over standardly used sequence-profile search methods (e.g. HMMER, PSI-BLAST) since it is particularly well-suited to discriminate proteins with variations inside conserved motifs (e.g. family subtypes) or motifs lacking essential residues (false positives, e.g. pseudoenzymes). RESULTS: In order to make mHMM widely accessible to a broader scientific community, we developed Leitmotif, an mHMM web application with many parametrization options easily accessible through intuitive interface. Substantial improvement of performance (ROC scores) was obtained by using two novel parameters. To the best of our knowledge, Leitmotif is the only available mHMM application. AVAILABILITY AND IMPLEMENTATION: Leitmotif is freely available at https://leitmotif.irb.hr. CONTACT: sinisa@heuristika.hr or ivan.vujaklija@fer.hr. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A Novel Alkaliphilic Streptomyces Inhibits ESKAPE Pathogens.

In an effort to stem the rising tide of multi-resistant bacteria, researchers have turned to niche environments in the hope of discovering new varieties of antibiotics. We investigated an ethnopharmacological (cure) from an alkaline/radon soil in the area of Boho, in the Fermanagh Scarplands (N. Ireland) for the presence of Streptomyces, a well-known producer of antibiotics. From this soil we isolated a novel (closest relative 57% of genome relatedness) Streptomyces sp. capable of growth at high alkaline pH (10.5) and tolerant of gamma radiation to 4 kGy. Genomic sequencing identified many alkaline tolerance (antiporter/multi-resistance) genes compared to S. coelicolor M145 (at 3:1), hence we designated the strain Streptomyces sp. myrophorea, isolate McG1, from the Greek, myro (fragrance) and phorea (porter/carrier). In vitro tests demonstrated the ability of the Streptomyces sp. myrophorea, isolate McG1 to inhibit the growth of many strains of ESKAPE pathogens; most notably carbapenem-resistant Acinetobacter baumannii (a critical pathogen on the WHO priority list of antibiotic-resistant bacteria), vancomycin-resistant Enterococcus faecium, and methicillin-resistant Staphylococcus aureus (both listed as high priority pathogens). Further in silico prediction of antimicrobial potential of Streptomyces sp. myrophorea, isolate McG1 by anti-SMASH and RAST software identified many secondary metabolite and toxicity resistance gene clusters (45 and 27, respectively) as well as many antibiotic resistance genes potentially related to antibiotic production. Follow-up in vitro tests show that the Streptomyces sp. myrophorea, isolate McG1 was resistant to 28 out of 36 clinical antibiotics. Although not a comprehensive analysis, we think that some of the Boho soils' reputed curative properties may be linked to the ability of Streptomyces sp. myrophorea, isolate McG1 to inhibit ESKAPE pathogens. More importantly, further analysis may elucidate other key components that could alleviate the tide of multi-resistant nosocomial infections.

3'-Terminated Overhangs Regulate DNA Double-Strand Break Processing in Escherichia coli.

Double-strand breaks (DSBs) are lethal DNA lesions, which are repaired by homologous recombination in Escherichia coli To study DSB processing in vivo, we induced DSBs into the E. coli chromosome by γ-irradiation and measured chromosomal degradation. We show that the DNA degradation is regulated by RecA protein concentration and its rate of association with single-stranded DNA (ssDNA). RecA decreased DNA degradation in wild-type, recB, and recD strains, indicating that it is a general phenomenon in E. coli On the other hand, DNA degradation was greatly reduced and unaffected by RecA in the recB1080 mutant (which produces long overhangs) and in a strain devoid of four exonucleases that degrade a 3' tail (ssExos). 3'-5' ssExos deficiency is epistatic to RecA deficiency concerning DNA degradation, suggesting that bound RecA is shielding the 3' tail from degradation by 3'-5' ssExos. Since 3' tail preservation is common to all these situations, we infer that RecA polymerization constitutes a subset of mechanisms for preserving the integrity of 3' tails emanating from DSBs, along with 3' tail's massive length, or prevention of their degradation by inactivation of 3'-5' ssExos. Thus, we conclude that 3' overhangs are crucial in controlling the extent of DSB processing in E. coli This study suggests a regulatory mechanism for DSB processing in E. coli, wherein 3' tails impose a negative feedback loop on DSB processing reactions, specifically on helicase reloading onto dsDNA ends.

Nucleolytic degradation of 3'-ending overhangs is essential for DNA-end resection in RecA-loading deficient recB mutants of Escherichia coli.

Degradation of a 5'-ending strand is the hallmark of the universal process of DNA double strand break (DSB) resection, which results in creation of the central recombination intermediate, a 3'-ending overhang. Here we show that in Escherichia coli recB1080/recB1067 mutants, which are devoid of RecBCD's nuclease and RecA loading activities, degradation of the unwound 3' tail is as essential as is degradation of its 5'-ending complement. Namely, a synergistic action of ExoI, ExoVII, SbcCD and ExoX single-strand specific exonucleases (ssExos) of 3'-5' polarity was essential for preserving cell viability, DNA repair and homologous recombination in the recB1080/recB1067 mutants, to the same extent as the redundant action of 5'-tail trimming ssExos RecJ and ExoVII. recB1080 derivatives lacking 3'-5' ssExos also showed a strong induction of the SOS response and greatly increased SOS-dependent mutagenesis. Furthermore, we show that ExoI and ExoVII ssExos act synergistically in suppressing illegitimate recombination in the recB1080 mutant but not in a wt strain, while working in concert with the RecQ helicase. Remarkably, 3'-5' ssExos show synergism with RecQ helicase in the recB1080 mutant in all the assays tested. The effect of inactivation of 3'-5' ssExos in the recB1080/recB1067 mutants was much stronger than in wt, recD, and recB strains. These results demonstrate that the presence of a long, reactive 3' overhang can be as toxic for a cell as its complete absence, i.e. it may prevent DSB repair. Our results indicate that coupling of helicase and RecA-loading activity during dsDNA-end resection is crucial in avoiding the deleterious effects of a long and stabile 3' tail in E. coli.

An effective approach for annotation of protein families with low sequence similarity and conserved motifs: identifying GDSL hydrolases across the plant kingdom.

BACKGROUND: The massive accumulation of protein sequences arising from the rapid development of high-throughput sequencing, coupled with automatic annotation, results in high levels of incorrect annotations. In this study, we describe an approach to decrease annotation errors of protein families characterized by low overall sequence similarity. The GDSL lipolytic family comprises proteins with multifunctional properties and high potential for pharmaceutical and industrial applications. The number of proteins assigned to this family has increased rapidly over the last few years. In particular, the natural abundance of GDSL enzymes reported recently in plants indicates that they could be a good source of novel GDSL enzymes. We noticed that a significant proportion of annotated sequences lack specific GDSL motif(s) or catalytic residue(s). Here, we applied motif-based sequence analyses to identify enzymes possessing conserved GDSL motifs in selected proteomes across the plant kingdom. RESULTS: Motif-based HMM scanning (Viterbi decoding-VD and posterior decoding-PD) and the here described PD/VD protocol were successfully applied on 12 selected plant proteomes to identify sequences with GDSL motifs. A significant number of identified GDSL sequences were novel. Moreover, our scanning approach successfully detected protein sequences lacking at least one of the essential motifs (171/820) annotated by Pfam profile search (PfamA) as GDSL. Based on these analyses we provide a curated list of GDSL enzymes from the selected plants. CLANS clustering and phylogenetic analysis helped us to gain a better insight into the evolutionary relationship of all identified GDSL sequences. Three novel GDSL subfamilies as well as unreported variations in GDSL motifs were discovered in this study. In addition, analyses of selected proteomes showed a remarkable expansion of GDSL enzymes in the lycophyte, Selaginella moellendorffii. Finally, we provide a general motif-HMM scanner which is easily accessible through the graphical user interface ( http://compbio.math.hr/ ). CONCLUSIONS: Our results show that scanning with a carefully parameterized motif-HMM is an effective approach for annotation of protein families with low sequence similarity and conserved motifs. The results of this study expand current knowledge and provide new insights into the evolution of the large GDSL-lipase family in land plants.

Integron diversity in marine environments.

Integrons are bacterial genetic elements known to be active vectors of antibiotic resistance among clinical bacteria. They are also found in bacterial communities from natural environments. Although integrons have become especially efficient for bacterial adaptation in the particular context of antibiotic usage, their role in natural environments in other contexts is still unknown. Indeed, most studies have focused on integrons and the spread of antibiotic resistance in freshwater or soil impacted by anthropogenic activities, with only few on marine environments. Notably, integrons show a wider diversity of both gene cassettes and integrase gene in natural environments than in clinical environments, suggesting a general role of integrons in bacterial adaptation. This article reviews the current knowledge on integrons in marine environments. We also present conclusions of our studies on polluted and nonpolluted backgrounds.

Exploring Actinobacteria assemblages in coastal marine sediments under contrasted Human influences in the West Istria Sea, Croatia.

The exploration of marine Actinobacteria has as major challenge to answer basic questions of microbial ecology that, in turn, will provide useful information to exploit Actinobacteria metabolisms in biotechnological processes. The ecological functions performed by Actinobacteria in marine sediments are still unclear and belongs to the most burning basic questions. The comparison of Actinobacteria communities inhabiting marine sediments that are under the influence of different contamination types will provide valuable information in the adaptation capacities of Actinobacteria to colonize specific ecological niche. In the present study, the characterization of different Actinobacteria assemblages according to contamination type revealed the ecological importance of Actinobacteria for maintaining both general biogeochemical functions through a "core" Actinobacteria community and specific roles associated with the presence of contaminants. Indeed, the results allowed to distinguish Actinobacteria genera and species operational taxonomic units (OTUs) able to cope with the presence of either (i) As, (ii) metals Ni, Fe, V, Cr, and Mn, or (iii) polycyclic aromatic hydrocarbons (PAHs) and toxic metals (Hg, Cd, Cu, Pb, and Zn). Such observations highlighted the metabolic capacities of Actinobacteria and their potential that should be taken into consideration and advantage during the implementation of bioremediation processes in marine ecosystems.

Structure-function relationships of two paralogous single-stranded DNA-binding proteins from Streptomyces coelicolor: implication of SsbB in chromosome segregation during sporulation.

The linear chromosome of Streptomyces coelicolor contains two paralogous ssb genes, ssbA and ssbB. Following mutational analysis, we concluded that ssbA is essential, whereas ssbB plays a key role in chromosome segregation during sporulation. In the ssbB mutant, ∼30% of spores lacked DNA. The two ssb genes were expressed differently; in minimal medium, gene expression was prolonged for both genes and significantly upregulated for ssbB. The ssbA gene is transcribed as part of a polycistronic mRNA from two initiation sites, 163 bp and 75 bp upstream of the rpsF translational start codon. The ssbB gene is transcribed as a monocistronic mRNA, from an unusual promoter region, 73 bp upstream of the AUG codon. Distinctive DNA-binding affinities of single-stranded DNA-binding proteins monitored by tryptophan fluorescent quenching and electrophoretic mobility shift were observed. The crystal structure of SsbB at 1.7 Šresolution revealed a common OB-fold, lack of the clamp-like structure conserved in SsbA and previously unpublished S-S bridges between the A/B and C/D subunits. This is the first report of the determination of paralogous single-stranded DNA-binding protein structures from the same organism. Phylogenetic analysis revealed frequent duplication of ssb genes in Actinobacteria, whereas their strong retention suggests that they are involved in important cellular functions.

Detecting posttranslational modifications of bacterial SSB proteins.

Posttranslational modifications of single-stranded DNA-binding proteins (SSBs), which are essential proteins in DNA metabolism, have been reported from prokaryotic to eukaryotic systems. While eukaryotic SSBs are regulated by phosphorylation on serine and threonine residues, bacterial SSB proteins are also phosphorylated on tyrosine residues. This was initially observed during a systematic search for global phosphotyrosine-containing proteins in Streptomyces, complex life cycle bacteria that support mycelial growth and spore formation. Tyrosine phosphorylation was further confirmed on SSB proteins from the spore-forming bacterium Bacillus subtilis and in the simpler prokaryote, Escherichia coli. However, a thorough study of this modification and its cognate kinase has been performed only on SSB proteins from Bacillus subtilis. It was shown that phosphorylation of B. subtilis SSB (SsbA) significantly increases binding affinity with single-stranded DNA in vitro. Mass spectrometry analysis of SsbA identified Tyr82 as the phosphorylation site. Analyses of the resolved and predicted crystal structures of SSB proteins from B. subtilis, E. coli, and S. coelicolor revealed that the Tyr phosphorylation site occupies similar positions in all three structures. Our results indicate that tyrosine phosphorylation of bacterial SSBs is a conserved modification in taxonomically distant bacteria.

Crystallization and preliminary X-ray diffraction studies of a complex of extracellular lipase from Streptomyces rimosus with the inhibitor 3,4-dichloroisocoumarin.

A recombinant lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) from the bacterium Streptomyces rimosus was inhibited by the serine protease inhibitor 3,4-dichloroisocoumarin and crystallized by the hanging-drop vapour-diffusion method at 291 K. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 38.1, b = 78.7, c = 56.6 Å, ß = 104.5° and probably two molecules in the asymmetric unit. Diffraction data were collected to 1.7 Å resolution using synchrotron radiation on the XRD beamline of the Elettra synchrotron, Trieste, Italy.

Clustering of protein domains for functional and evolutionary studies.

BACKGROUND: The number of protein family members defined by DNA sequencing is usually much larger than those characterised experimentally. This paper describes a method to divide protein families into subtypes purely on sequence criteria. Comparison with experimental data allows an independent test of the quality of the clustering. RESULTS: An evolutionary split statistic is calculated for each column in a protein multiple sequence alignment; the statistic has a larger value when a column is better described by an evolutionary model that assumes clustering around two or more amino acids rather than a single amino acid. The user selects columns (typically the top ranked columns) to construct a motif. The motif is used to divide the family into subtypes using a stochastic optimization procedure related to the deterministic annealing EM algorithm (DAEM), which yields a specificity score showing how well each family member is assigned to a subtype. The clustering obtained is not strongly dependent on the number of amino acids chosen for the motif. The robustness of this method was demonstrated using six well characterized protein families: nucleotidyl cyclase, protein kinase, dehydrogenase, two polyketide synthase domains and small heat shock proteins. Phylogenetic trees did not allow accurate clustering for three of the six families. CONCLUSION: The method clustered the families into functional subtypes with an accuracy of 90 to 100%. False assignments usually had a low specificity score.

Structure of the single-stranded DNA-binding protein from Streptomyces coelicolor.

The crystal structure of the single-stranded DNA-binding protein (SSB) from Streptomyces coelicolor, a filamentous soil bacterium with a complex life cycle and a linear chromosome, has been solved and refined at 2.1 A resolution. The three-dimensional structure shows a common conserved central OB-fold that is found in all structurally determined SSB proteins. However, it shows variations in quaternary structure that have previously only been found in mycobacterial SSBs. The strand involved in the clamp mechanism characteristic of this type of quaternary structure leads to higher stability of the homotetramer. To the best of our knowledge, this is the first X-ray structure of an SSB protein from a member of the genus Streptomyces and it was predicted to be the most stable of the structurally characterized bacterial or human mitochondrial SSBs.

The SGNH-hydrolase of Streptomyces coelicolor has (aryl)esterase and a true lipase activity.

The Streptomyces coelicolor A3(2) gene SCI11.14c was overexpressed and purified as a His-tagged protein from heterologous host, Streptomyces lividans. The purification procedure resulted in 34.1-fold increase in specific activity with an overall yield of 21.4%. Biochemical and physical properties of the purified enzyme were investigated and it was shown that it possesses (aryl)esterase and a true lipase activity. The enzyme was able to hydrolyze p-nitrophenyl-, alpha- and beta-naphthyl esters and poly(oxyethylene) sorbitan monoesters (Tween 20-80). It showed pronounced activity towards p-nitrophenyl and alpha- and beta-naphthyl esters of C(12)-C(16). Higher activity was observed with alpha-naphthyl esters. The enzyme hydrolyzed triolein (specific activity: 91.9 U/mg) and a wide range of oils with a preference for those having higher content of linoleic or oleic acid (C18:2; C18:1, cis). The active-site serine specific inhibitor 3,4-dichloroisocoumarin (DCI) strongly inhibited the enzyme, while tetrahydrofurane and 1,4-dioxane significantly increased (2- and 4- fold, respectively) hydrolytic activity of lipase towards p-nitrophenyl caprylate. The enzyme exhibited relatively high temperature optimum (55 degrees C) and thermal stability. CD analysis revealed predominance of alpha-helical structure (54% alpha-helix, 21% beta-sheet) and a T(m) value at 66 degrees C. Systematic bioinformatic analysis of deduced amino acid sequence of S. coelicolor enzyme placed it to the SGNH-hydrolase family. Phylogenetic analysis of the predicted protein homologous to the S. coelicolor SGNH-hydrolase generated three distinct groups consisting of proteins from Actinomycetales, Ascomycota and Nematoda. At present it seems that these enzymes are most conserved among soil inhabiting organisms.

Proposed arrangement of proteins forming a bacterial type II polyketide synthase.

Aklanonic acid is synthesized by a type II polyketide synthase (PKS) composed of eight protein subunits. The network of protein interactions within this complex was investigated using a yeast two-hybrid system, by coaffinity chromatography and by two different computer-aided protein docking simulations. Results suggest that the ketosynthase (KS) alpha and beta subunits interact with each other, and that the KSalpha subunit also probably interacts with a malonyl-CoA:ACP acyltransferase (DpsD), forming a putative minimal synthase. We speculate that DpsD may physically inhibit the priming reaction, allowing the choice of propionate rather than acetate as the starter unit. We also suggest a structural role for the cyclase (DpsY) in maintaining the overall structural integrity of the complex.

Functional tyrosine residue in the active center of human dipeptidyl peptidase III.

Abstract Human dipeptidyl peptidase III (DPP III) is a member of the metallopeptidase family M49 with an implied role in the pain-modulatory system and endogenous defense against oxidative stress. Here, we report the heterologous expression of human DPP III and the site-directed mutagenesis results which demonstrate a functional role for Tyr318 at the active site of this enzyme. The substitution of Tyr318 to Phe decreased kcat by two orders of magnitude without altering the binding affinity of substrate, or of a competitive hydroxamate inhibitor designed to interact with S1 and S2 subsites. The results indicate that the conserved tyrosine could be involved in transition state stabilization during the catalytic action of M49 peptidases.