Genome Mining of Linaridins Provides Insights into the Widely Distributed LinC Oxidoreductases.

Linaridins are a family of underexplored ribosomally synthesized and post-translationally modified peptides despite the prevalence of their biosynthetic gene clusters (BGCs) in microbial genomes, as shown by bioinformatic studies. Our genome mining efforts reveal that 96 putative oxidoreductase genes, namely, LinC, are encoded in linaridin BGCs. We heterologously expressed two such LinC-containing linaridin BGCs, yan and ydn, from Streptomyces yunnanensis and obtained three new linaridins, named yunnanaridins A-C (1-3). Their structures are characterized by Z-configurations of the dehydrobutyrines and the presence of a variety of epimerized amino acid residues. Yunnanaridin A (1) is the sixth member of the family of type-B linaridins, whereas yunnanaridins B (2) and C (3) represent the first examples of expressed type-C linaridins. Interestingly, heterologous expression of the same BGCs with LinC in-frame knockouts produced the same compounds. This work expands the structural diversity of linaridins and provides evidence for the notion that the widespread LinCs may not be involved in linaridin biosynthesis.

Lanthipeptides from the Same Core Sequence: Characterization of a Class II Lanthipeptide Synthetase from Microcystis aeruginosa NIES-88.

Class II lanthipeptide synthetases (LanMs) are relatively promiscuous to core peptide variations. Previous studies have shown that different LanMs catalyze identical reactions on the same core sequence fused to their respective cognate leaders. We characterized a new LanM enzyme from Microcystis aeruginosa NIES-88, MalM, and demonstrated that MalM and ProcM exhibited disparate dehydration and cyclization patterns on identical core peptides. Our study provided new insights into the regioselectivity of LanMs and showcased an appropriate strategy for lanthipeptide structural diversity engineering.

Functional elucidation of TfuA in peptide backbone thioamidation.

YcaO enzymes catalyze several post-translational modifications on peptide substrates, including thioamidation, which substitutes an amide oxygen with sulfur. Most predicted thioamide-forming YcaO enzymes are encoded adjacent to TfuA, which when present, is required for thioamidation. While activation of the peptide amide backbone is well established for YcaO enzymes, the function of TfuA has remained enigmatic. Here we characterize the TfuA protein involved in methyl-coenzyme M reductase thioamidation and demonstrate that TfuA catalyzes the hydrolysis of thiocarboxylated ThiS (ThiS-COSH), a proteinaceous sulfur donor, and enhances the affinity of YcaO toward the thioamidation substrate. We also report a crystal structure of a TfuA, which displays a new protein fold. Our structural and mutational analyses of TfuA have uncovered conserved binding interfaces with YcaO and ThiS in addition to revealing a hydrolase-like active site featuring a Ser-Lys catalytic pair.

Structure-Guided Biochemical Analysis of Quorum Signal Synthase Specificities.

Many bacteria use membrane-diffusible small molecule quorum signals to coordinate gene transcription in response to changes in cell density, known as quorum sensing (QS). Among these, acyl-homoserine lactones (AHL) are widely distributed in Proteobacteria and are involved in controlling the expression of virulence genes and biofilm formation in pathogens, such as Pseudomonas aeruginosa. AHL molecules are specifically biosynthesized by the cognate LuxI type AHL synthases using S-adenosylmethionine (SAM) and either acyl carrier protein (ACP)- or CoA-coupled fatty acids through a two-step reaction. Here, we characterize a CoA-dependent LuxI synthase from Rhodopseudomonas palustris that utilizes an aryl-CoA substrate that is environmentally derived, specifically p-coumaric acid. We leverage structures of this aryl-CoA-dependent synthase, along with our prior studies of an acyl-CoA-dependent synthase, to identify residues that confer substrate chain specificity in these enzymes. We test our predictions by carrying out biochemical, kinetic, and structural characterization of representative AHL signal synthases. Our studies provide an understanding of various AHL synthases that may be deployed in synthetic biological applications and inform on the design of specific small molecule therapeutics that can restrict virulence by targeting quorum signaling.

Functional interactions between posttranslationally modified amino acids of methyl-coenzyme M reductase in Methanosarcina acetivorans.

The enzyme methyl-coenzyme M reductase (MCR) plays an important role in mediating global levels of methane by catalyzing a reversible reaction that leads to the production or consumption of this potent greenhouse gas in methanogenic and methanotrophic archaea. In methanogenic archaea, the alpha subunit of MCR (McrA) typically contains four to six posttranslationally modified amino acids near the active site. Recent studies have identified enzymes performing two of these modifications (thioglycine and 5-[S]-methylarginine), yet little is known about the formation and function of the remaining posttranslationally modified residues. Here, we provide in vivo evidence that a dedicated S-adenosylmethionine-dependent methyltransferase encoded by a gene we designated methylcysteine modification (mcmA) is responsible for formation of S-methylcysteine in Methanosarcina acetivorans McrA. Phenotypic analysis of mutants incapable of cysteine methylation suggests that the S-methylcysteine residue might play a role in adaption to mesophilic conditions. To examine the interactions between the S-methylcysteine residue and the previously characterized thioglycine, 5-(S)-methylarginine modifications, we generated M. acetivorans mutants lacking the three known modification genes in all possible combinations. Phenotypic analyses revealed complex, physiologically relevant interactions between the modified residues, which alter the thermal stability of MCR in a combinatorial fashion that is not readily predictable from the phenotypes of single mutants. High-resolution crystal structures of inactive MCR lacking the modified amino acids were indistinguishable from the fully modified enzyme, suggesting that interactions between the posttranslationally modified residues do not exert a major influence on the static structure of the enzyme but rather serve to fine-tune the activity and efficiency of MCR.

Recent Advances in the Discovery and Biosynthetic Study of Eukaryotic RiPP Natural Products.

Natural products have played indispensable roles in drug development and biomedical research. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a group of fast-expanding natural products attribute to genome mining efforts in recent years. Most RiPP natural products were discovered from bacteria, yet many eukaryotic cyclic peptides turned out to be of RiPP origin. This review article presents recent advances in the discovery of eukaryotic RiPP natural products, the elucidation of their biosynthetic pathways, and the molecular basis for their biosynthetic enzyme catalysis.

Molecular basis for the substrate specificity of quorum signal synthases.

In several Proteobacteria, LuxI-type enzymes catalyze the biosynthesis of acyl-homoserine lactones (AHL) signals using S-adenosyl-l-methionine and either cellular acyl carrier protein (ACP)-coupled fatty acids or CoA-aryl/acyl moieties as progenitors. Little is known about the molecular mechanism of signal biosynthesis, the basis for substrate specificity, or the rationale for donor specificity for any LuxI member. Here, we present several cocrystal structures of BjaI, a CoA-dependent LuxI homolog that represent views of enzyme complexes that exist along the reaction coordinate of signal synthesis. Complementary biophysical, structure-function, and kinetic analysis define the features that facilitate the unusual acyl conjugation with S-adenosylmethionine (SAM). We also identify the determinant that establishes specificity for the acyl donor and identify residues that are critical for acyl/aryl specificity. These results highlight how a prevalent scaffold has evolved to catalyze quorum signal synthesis and provide a framework for the design of small-molecule antagonists of quorum signaling.

The pimeloyl-CoA synthetase BioW defines a new fold for adenylate-forming enzymes.

Reactions that activate carboxylates through acyl-adenylate intermediates are found throughout biology and include acyl- and aryl-CoA synthetases and tRNA synthetases. Here we describe the characterization of Aquifex aeolicus BioW, which represents a new protein fold within the superfamily of adenylating enzymes. Substrate-bound structures identified the enzyme active site and elucidated the mechanistic strategy for conjugating CoA to the seven-carbon α,ω-dicarboxylate pimelate, a biotin precursor. Proper position of reactive groups for the two half-reactions is achieved solely through movements of active site residues, as confirmed by site-directed mutational analysis. The ability of BioW to hydrolyze adenylates of noncognate substrates is reminiscent of pre-transfer proofreading observed in some tRNA synthetases, and we show that this activity can be abolished by mutation of a single residue. These studies illustrate how BioW can carry out three different biologically prevalent chemical reactions (adenylation, thioesterification, and proofreading) in the context of a new protein fold.

Trichinenlides A-T, mexicanolide-type limonoids from Trichilia sinensis.

Twenty new mexicanolide-type limonoids, namely, trichinenlides A-T (1-20), and 11 known analogues were isolated from the leaves and twigs of Trichilia sinensis. Trichinenlides B (2) and C (3) and heytrijunolide D exhibited inhibition against lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages with IC50 values of 2.85, 1.88, and 3.33 µM, respectively.

Chemical constituents from Brucea javanica.

Fourteen apotirucallane-type triterpenoids, named brujavanones A-N, were isolated from the twigs of Brucea javanica, along with four known quassinoids and seven known lignans from the seeds of B. javanica. Their structures were elucidated on the basis of extensive spectroscopic data analysis. The structure of a previously reported triterpenoid, bruceajavanin C, was revised as its C-21 epimer. The cytotoxic activities of triterpenoids and quassinoids against two human tumor cell lines, HL-60 and A-549, were evaluated, but all the compounds were inactive (IC50>10 µM).

Sesquiterpenes from Dysoxylum oliganthum and Dysoxylum excelsum.

Three new sesquiterpenes (1-3), (6R,7S,11R,10S)-15-hydroxy-sesquisabinene hydrate (1), (6R,7R,11S,10S)-15-hydroxy-sesquisabinene hydrate (2), and (6R,7R,10S)-15-hydroxy-zingiberenol (3), along with three known compounds, were isolated from the stems of Dysoxylum oliganthum; and three new isodaucane (salvionane) sesquiterpenes, namely isodauc-6-ene-10ß,14-diol (4), 4-epi-isodauc-6-ene-10ß,14-diol (5), and 4-epi-6α,10ß-dihydroxy-artabotrol (6) together with 15 known compounds were isolated from the twigs and leaves of D. excelsum. Their structures were established on the basis of extensive spectroscopic analysis and chemical shifts. The absolute configuration of C-10 in compounds 1-3 of a rare class was determined by using Snatzke's method.

Constituents of Trigonostemon heterophyllus.

Six new diterpenoids, 1-6, a new prenylated sesquiterpenoid, 7, and six known compounds were isolated from Trigonostemon heterophyllus. The structures of 1-7 were elucidated on the basis of NMR and MS analysis. The daphnane diterpenoids trigoheterins A (1) and B (2) possess a rare 4,6-oxetane moiety within this compound class, and trigoheteric acid methyl ester (6) is the first example of a 15,16-dinor-3,4-seco-cleistanthane. Trigoheterin E (5) exhibited cytotoxic activity against the HL-60 (IC50) 1.8 µM) and A-549 (IC50 10.0 µM) human cancer cell lines.

Angustimine and angustifolimine: two new alkaloids from Daphniphyllum angustifolium.

Two new alkaloids, angustimine (1) featuring an unprecedented skeleton, and angustifolimine (2) being a rare diamino Daphniphyllum alkaloid, were isolated from Daphniphyllum angustifolium. Their structures were elucidated by extensive spectroscopic analysis. The biosynthetic origin of 1 was postulated. Cytotoxic activities of 1 and 2 were tested, and all of them were inactive.

Daphnane-type diterpenoids from Trigonostemon howii.

Nine new daphnane-type diterpenoids (1-9), named trigohownins A-I, and four known analogues were isolated from Trigonostemon howii. Their structures were elucidated on the basis of extensive NMR and MS analyses. Trigohownins A (1) and D (4) exhibited moderate cytotoxic activity against the HL-60 tumor cell line, with IC50 values of 17.0 and 9.3 µM, respectively.