Mechanistic analysis of nonribosomal peptide synthetases

Considering the ongoing rise of the multidrug-resistant bacterial infections, it is essential to expand the available repertoire of therapeutic agents. Microbial natural products are an indispensable source of novel activities and continue to serve as our main provider of antibiotics and chemotherapeutics. Nonribosomal peptides are among the most widespread natural products in bacteria and fungi. Their importance is best illustrated by their complexity and the amounts of resources dedicated to building the underlying biosynthetic machineries nonribosomal peptide synthetases (NRPS). These gigantic, multidomain enzymes synthesize peptides by linking individual amino acid units in an assembly line fashion. Six decades of NRPS research have resulted in several remarkable tailoring successes. However, the lack of mechanistic understanding of the inner workings of NRPSs has prevented the development of a general workflow which would reliably generate functional enzymes and new drugs. Aspiring to alleviate these obstacles, this thesis offers critical insights into adenylation and the interplay with condensation, two fundamental NRPS reactions

Specificity prediction of adenylation domains in nonribosomal peptide synthetases (NRPS) using transductive support vector machines (TSVMs)

We present a new support vector machine (SVM)-based approach to predict the substrate specificity of subtypes of a given protein sequence family. We demonstrate the usefulness of this method on the example of aryl acid-activating and amino acid-activating adenylation domains (A domains) of nonribosomal peptide synthetases (NRPS). The residues of gramicidin synthetase A that are 8 Å around the substrate amino acid and corresponding positions of other adenylation domain sequences with 397 known and unknown specificities were extracted and used to encode this physico-chemical fingerprint into normalized real-valued feature vectors based on the physico-chemical properties of the amino acids. The SVM software package SVM(light) was used for training and classification, with transductive SVMs to take advantage of the information inherent in unlabeled data. Specificities for very similar substrates that frequently show cross-specificities were pooled to the so-called composite specificities and predictive models were built for them. The reliability of the models was confirmed in cross-validations and in comparison with a currently used sequence-comparison-based method. When comparing the predictions for 1230 NRPS A domains that are currently detectable in UniProt, the new method was able to give a specificity prediction in an additional 18% of the cases compared with the old method. For 70% of the sequences both methods agreed, for <6% they did not, mainly on low-confidence predictions by the existing method. None of the predictive methods could infer any specificity for 2.4% of the sequences, suggesting completely new types of specificity

Numerical and Evolutionary Optimization 2020

This book was established after the 8th International Workshop on Numerical and Evolutionary Optimization (NEO), representing a collection of papers on the intersection of the two research areas covered at this workshop: numerical optimization and evolutionary search techniques. While focusing on the design of fast and reliable methods lying across these two paradigms, the resulting techniques are strongly applicable to a broad class of real-world problems, such as pattern recognition, routing, energy, lines of production, prediction, and modeling, among others. This volume is intended to serve as a useful reference for mathematicians, engineers, and computer scientists to explore current issues and solutions emerging from these mathematical and computational methods and their applications

Algorithm for backrub motions in protein design

Motivation: The Backrub is a small but kinematically efficient side-chain-coupled local backbone motion frequently observed in atomic-resolution crystal structures of proteins. A backrub shifts the Cα–Cβ orientation of a given side-chain by rigid-body dipeptide rotation plus smaller individual rotations of the two peptides, with virtually no change in the rest of the protein. Backrubs can therefore provide a biophysically realistic model of local backbone flexibility for structure-based protein design. Previously, however, backrub motions were applied via manual interactive model-building, so their incorporation into a protein design algorithm (a simultaneous search over mutation and backbone/side-chain conformation space) was infeasible

Identifying the source of unknown microcystin genes and predicting microcystin variants by comparing genes within uncultured cyanobacterial cells

While multiple phylogenetic markers have been used in the culture independent study of microcystin producing cyanobacteria, in only a few instances have multiple markers been studied within individual cells, and in all cases these studies have been conducted with cultured isolates. Here, we isolate and evaluate large DNA fragments (\u3e 6 kb) encompassing two genes involved in microcystin biosynthesis (mcyA2 and mcyB1) and use them to identify the source of gene fragments found in water samples. Further investigation of these gene loci from individual cyanobacterial cells allowed for improved analysis of the genetic diversity within microcystin producers as well as a method to predict microcystin variants for individuals. These efforts have also identified the source of the novel mcyA genotype previously termed Microcystis-like that is pervasive in the Laurentian Great Lakes and predict the microcystin variant(s) that it produces

Recurrent adenylation domain replacement in the microcystin synthetase gene cluster

<p>Abstract</p> <p>Background</p> <p>Microcystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. Microcystins are synthesized on large NRPS-PKS enzyme complexes. Many structural variants of microcystins are produced simulatenously. A recombination event between the first module of <it>mcyB (mcyB1) </it>and <it>mcyC </it>in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus <it>Microcystis</it>.</p> <p>Results</p> <p>Here we undertook a phylogenetic study to investigate the order and timing of recombination between the <it>mcyB1 </it>and <it>mcyC </it>genes in a diverse selection of microcystin producing cyanobacteria. Our results provide support for complex evolutionary processes taking place at the <it>mcyB1 </it>and <it>mcyC </it>adenylation domains which recognize and activate the amino acids found at X and Z positions. We find evidence for recent recombination between <it>mcyB1 </it>and <it>mcyC </it>in strains of the genera <it>Anabaena</it>, <it>Microcystis</it>, and <it>Hapalosiphon</it>. We also find clear evidence for independent adenylation domain conversion of <it>mcyB1 </it>by unrelated peptide synthetase modules in strains of the genera <it>Nostoc </it>and <it>Microcystis</it>. The recombination events replace only the adenylation domain in each case and the condensation domains of <it>mcyB1 </it>and <it>mcyC </it>are not transferred together with the adenylation domain. Our findings demonstrate that the <it>mcyB1 </it>and <it>mcyC </it>adenylation domains are recombination hotspots in the microcystin synthetase gene cluster.</p> <p>Conclusion</p> <p>Recombination is thought to be one of the main mechanisms driving the diversification of NRPSs. However, there is very little information on how recombination takes place in nature. This study demonstrates that functional peptide synthetases are created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains.</p

Recombination and selectional forces in cyanopeptolin NRPS operons from highly similar, but geographically remote Planktothrix strains

<p>Abstract</p> <p>Background</p> <p>Cyanopeptolins are nonribosomally produced heptapetides showing a highly variable composition. The cyanopeptolin synthetase operon has previously been investigated in three strains from the genera <it>Microcystis</it>, <it>Planktothrix </it>and <it>Anabaena</it>. Cyanopeptolins are displaying protease inhibitor activity, but the biological function(s) is (are) unknown. Cyanopeptolin gene cluster variability and biological functions of the peptide variants are likely to be interconnected.</p> <p>Results</p> <p>We have investigated two cyanopeptolin gene clusters from highly similar, but geographically remote strains of the same genus. Sequencing of a nonribosomal peptide synthetase (NRPS) cyanopeptolin gene cluster from the Japanese strain <it>Planktothrix </it>NIES 205 (205-<it>oci</it>), showed the 30 kb gene cluster to be highly similar to the <it>oci </it>gene cluster previously described in <it>Planktothrix </it>NIVA CYA 116, isolated in Norway. Both operons contained seven NRPS modules, a sulfotransferase (S) and a glyceric acid loading (GA)-domain. Sequence analyses showed a high degree of conservation, except for the presence of an epimerase domain in NIES 205 and the regions around the epimerase, showing high substitution rates and Ka/Ks values above 1. The two strains produce almost identical cyanopeptolins, cyanopeptolin-1138 and oscillapeptin E respectively, but with slight differences regarding the production of minor cyanopeptolin variants. These variants may be the result of relaxed adenylation (A)-domain specificity in the nonribosomal enzyme complex. Other genetic markers (16S rRNA, <it>ntc</it>A and the phycocyanin <it>cpc</it>BA spacer) were identical, supporting that these geographically separated <it>Planktothrix </it>strains are closely related.</p> <p>Conclusion</p> <p>A horizontal gene transfer event resulting in exchange of a whole module-encoding region was observed. Nucleotide statistics indicate that both purifying selection and positive selection forces are operating on the gene cluster. The positive selection forces are acting within and around the epimerase insertion while purifying selection conserves the remaining (major) part of the gene cluster. The presence of an epimerase in the gene cluster is in line with the D-configuration of Htyr, determined experimentally in oscillapeptin E in a previous study.</p

SBSPKS: structure based sequence analysis of polyketide synthases

Polyketide synthases (PKSs) catalyze biosynthesis of a diverse family of pharmaceutically important secondary metabolites. Bioinformatics analysis of sequence and structural features of PKS proteins plays a crucial role in discovery of new natural products by genome mining, as well as in design of novel secondary metabolites by biosynthetic engineering. The availability of the crystal structures of various PKS catalytic and docking domains, and mammalian fatty acid synthase module prompted us to develop SBSPKS software which consists of three major components. Model_3D_PKS can be used for modeling, visualization and analysis of 3D structure of individual PKS catalytic domains, dimeric structures for complete PKS modules and prediction of substrate specificity. Dock_Dom_Anal identifies the key interacting residue pairs in inter-subunit interfaces based on alignment of inter-polypeptide linker sequences to the docking domain structure. In case of modular PKS with multiple open reading frames (ORFs), it can predict the cognate order of substrate channeling based on combinatorial evaluation of all possible interface contacts. NRPS–PKS provides user friendly tools for identifying various catalytic domains in the sequence of a Type I PKS protein and comparing them with experimentally characterized PKS/NRPS clusters cataloged in the backend databases of SBSPKS. SBSPKS is available at http://www.nii.ac.in/sbspks.html