Cyclic dipeptides and the human microbiome : opportunities and challenges

Funding: C.M.C. is funded by the Wellcome Trust (210486/Z/18/Z and [204821/Z/16/Z] to the University of St Andrews). C.E.O. is the recipient of a Carnegie Trust PhD studentship (PHD008520).Research into the human microbiome has implicated its constituents in a variety of non-communicable diseases, with certain microbes found to promote health and others leading to dysbiosis and pathogenesis. Microbes communicate and coordinate their behaviour through the secretion of small molecules, such as cyclic dipeptides (CDPs) into their surrounding environment. CDPs are ubiquitous signalling molecules that exhibit a wide range of biological activities, with particular relevance to human health due to their potential to act as microbiome modulators.Publisher PDFPeer reviewe

OLAVO AMARAL E A DIFICULDADE DE NOS COMUNICARMOS NO MUNDO ATUAL

Resenha do livro "Dicionário de línguas imaginárias", do escritor Olavo Amaral, abordando as narrativas contidas no livro, bem como a maneira com que elas tratam a dificuldade de comunicação humana no mundo atual

Mechanisms of cyanobactin biosynthesis

This work was supported by the European Research Council (339367), UK Biotechnology and Biological Sciences Research Council (K015508/1).Cyanobactins are a diverse collection of natural products that originate from short peptides made on a ribosome. The amino acids are modified in a series of transformations catalyzed by multiple enzymes. The patellamide pathway is the most well studied and characterized example. Here we review the structures and mechanisms of the enzymes that cleave peptide bonds, macrocyclise peptides, heterocyclise cysteine (as well as threonine and serine) residues, oxidize five-membered heterocycles and attach prenyl groups. Some enzymes operate by novel mechanisms which is of interest and in addition the enzymes uncouple recognition from catalysis. The normally tight relationship between these factors hinders biotechnology. The cyanobactin pathway may be particularly suitable for exploitation, with progress observed with in vivo and in vitro approaches.PostprintPeer reviewe

Unveiling the catalytic mechanism of a processive metalloaminopeptidase

Funding: C.M.C. is funded by the Wellcome Trust (210486/Z/18/Z and [204821/Z/16/Z] to the University of StAndrews). M.C.S.is funded by a PhD studentship from the University of St Andrews. B.E.B. acknowledges equipment funding by BBSRC (BB/R013780/1).Intracellular leucine aminopeptidases (PepA) are metalloproteases from the family M17. These enzymes catalyze peptide bond cleavage, removing N-terminal residues from peptide and protein substrates, with consequences for protein homeostasis and quality control. While general mechanistic studies using model substrates have been conducted on PepA enzymes from various organisms, specific information about their substrate preferences and promiscuity, choice of metal, activation mechanisms, and the steps that limit steady-state turnover remain unexplored. Here, we dissected the catalytic and chemical mechanisms of PaPepA: a leucine aminopeptidase from Pseudomonas aeruginosa. Cleavage assays using peptides and small-molecule substrate mimics allowed us to propose a mechanism for catalysis. Steady-state and pre-steady-state kinetics, pH rate profiles, solvent kinetic isotope effects, and biophysical techniques were used to evaluate metal binding and activation. This revealed that metal binding to a tight affinity site is insufficient for enzyme activity; binding to a weaker affinity site is essential for catalysis. Progress curves for peptide hydrolysis and crystal structures of free and inhibitor-bound PaPepA revealed that PaPepA cleaves peptide substrates in a processive manner. We propose three distinct modes for activity regulation: tight packing of PaPepA in a hexameric assembly controls substrate length and reaction processivity; the product leucine acts as an inhibitor, and the high concentration of metal ions required for activation limits catalytic turnover. Our work uncovers catalysis by a metalloaminopeptidase, revealing the intricacies of metal activation and substrate selection. This will pave the way for a deeper understanding of metalloenzymes and processive peptidases/proteases.Publisher PDFPeer reviewe

Active site remodelling of a cyclodipeptide synthase redefines substrate scope

Funding: Wellcome Trust (210486/Z/18/Z), Cunningham Trust (PhD-CT-18-41).Cyclodipeptide synthases (CDPSs) generate a wide range of cyclic dipeptides using aminoacylated tRNAs as substrates. Histidine-containing cyclic dipeptides have important biological activities as anticancer and neuroprotective molecules. Out of the 120 experimentally validated CDPS members, only two are known to accept histidine as a substrate yielding cyclo(His-Phe) and cyclo(His-Pro) as products. It is not fully understood how CDPSs select their substrates, and we must rely on bioprospecting to find new enzymes and novel bioactive cyclic dipeptides. Here, we developed an in vitro system to generate an extensive library of molecules using canonical and non-canonical amino acids as substrates, expanding the chemical space of histidine-containing cyclic dipeptide analogues. To investigate substrate selection we determined the structure of a cyclo(His-Pro)-producing CDPS. Three consecutive generations harbouring single, double and triple residue substitutions elucidated the histidine selection mechanism. Moreover, substrate selection was redefined, yielding enzyme variants that became capable of utilising phenylalanine and leucine. Our work successfully engineered a CDPS to yield different products, paving the way to direct the promiscuity of these enzymes to produce molecules of our choosing.Publisher PDFPeer reviewe

Characterization of a dual function macrocyclase enables design and use of efficient macrocyclization substrates

H.L. is funded by the George & Stella Lee Scholarship and Criticat EPSRC. This project was also funded by the European Research Council project 339367 NCB-TNT and by the BBSRC (K015508/1). JHN is 1000 talent scholar of the Chinese Academy of Sciences at the University of Sichuan.Peptide macrocycles are promising therapeutic molecules because they are protease resistant, structurally rigid, membrane permeable and capable of modulating protein-protein interactions. Here, we report the characterization of the dual function macrocyclase-peptidase enzyme involved in the biosynthesis of the highly toxic Amanitin toxin family of macrocycles. The enzyme first removes 10 residues from the N-terminus of a 35-residue substrate. Conformational trapping of the amino acid peptide forces the enzyme to release this intermediate rather than proceed to macrocyclization. The enzyme rebinds the 25 amino acid peptide in a different conformation and catalyzes macrocyclization of the N-terminal 8 residues. Structures of the enzyme bound to both substrates and biophysical analysis characterize the different binding modes rationalizing the mechanism. Using these insights simpler substrates with only five C-terminal residues were designed, allowing the enzyme to be more effectively exploited in biotechnology.Publisher PDFPeer reviewe

An anti-biofilm cyclic peptide targets a secreted aminopeptidase from P. aeruginosa

Funding: CMC and CJH are funded by the Wellcome Trust (210486/Z/18/Z), additional funding was provided by the University of St Andrews Impact Innovation Fund. MB (Bergkessel) is funded by the University of Dundee/Wellcome Trust Institutional Strategic Support Fund [204816/Z/16/Z] and UKRI Future Leaders Fellowship (funded by the Medical Research Council) [MR/T041811/1].Pseudomonas aeruginosa is an opportunistic pathogen that causes serious illness, especially in immunocompromised individuals. P. aeruginosa forms biofilms that contribute to growth and persistence in a wide range of environments. Here we investigated the aminopeptidase, P. aeruginosa aminopeptidase (PaAP) from P. aeruginosa, which is highly abundant in the biofilm matrix. PaAP is associated with biofilm development and contributes to nutrient recycling. We confirmed that post-translational processing was required for activation and PaAP is a promiscuous aminopeptidase acting on unstructured regions of peptides and proteins. Crystal structures of wild-type enzymes and variants revealed the mechanism of autoinhibition, whereby the C-terminal propeptide locks the protease-associated domain and the catalytic peptidase domain into a self-inhibited conformation. Inspired by this, we designed a highly potent small cyclic-peptide inhibitor that recapitulates the deleterious phenotype observed with a PaAP deletion variant in biofilm assays and present a path toward targeting secreted proteins in a biofilm context.Publisher PDFPeer reviewe

Snapshots of the reaction coordinate of a thermophilic 2'-deoxyribonucleoside/ribonucleoside transferase

Funding: P.T. is funded by IBioIC (IBioIC 2020-2-1), and C.M.C. is funded by the Wellcome Trust (217078/Z/19/Z). C.M.C. and D.H. are funded by research grants from NuCana plc..Nucleosides are ubiquitous to life and are required for the synthesis of DNA, RNA, and other molecules crucial for cell survival. Despite the notoriously difficult organic synthesis of nucleosides, 2′-deoxynucleoside analogues can interfere with natural DNA replication and repair and are successfully employed as anticancer, antiviral, and antimicrobial compounds. Nucleoside 2′-deoxyribosyltransferase (dNDT) enzymes catalyze transglycosylation via a covalent 2′-deoxyribosylated enzyme intermediate with retention of configuration, having applications in the biocatalytic synthesis of 2′-deoxynucleoside analogues in a single step. Here, we characterize the structure and function of a thermophilic dNDT, the protein from Chroococcidiopsis thermalis (CtNDT). We combined enzyme kinetics with structural and biophysical studies to dissect mechanistic features in the reaction coordinate, leading to product formation. Bell-shaped pH-rate profiles demonstrate activity in a broad pH range of 5.5–9.5, with two very distinct pKa values. A pronounced viscosity effect on the turnover rate indicates a diffusional step, likely product (nucleobase1) release, to be rate-limiting. Temperature studies revealed an extremely curved profile, suggesting a large negative activation heat capacity. We trapped a 2′-fluoro-2′-deoxyarabinosyl-enzyme intermediate by mass spectrometry and determined high-resolution structures of the protein in its unliganded, substrate-bound, ribosylated, 2′-difluoro-2′-deoxyribosylated, and in complex with probable transition-state analogues. We reveal key features underlying (2′-deoxy)ribonucleoside selection, as CtNDT can also use ribonucleosides as substrates, albeit with a lower efficiency. Ribonucleosides are the building blocks of RNA and other key intracellular metabolites participating in energy and metabolism, expanding the scope of use of CtNDT in biocatalysis.Peer reviewe

Considerações para implementação de ferramentas multiplataforma para monitramento de sistemas virtualizados

A virtualização através do uso de máquinas virtuais sobre máquinas físicas, para executar diferentes sistemas em diferentes domínios de aplicação tem sido uma abordagem comumente adotada em diferentes contextos. A possibilidade de se abstrair plataformas, infraestrutura ou software como um serviço passou a ser uma técnica válida para executar sistemas usando Computação na Nuvem (Cloud Computing) onde a virtualização é uma das principais tecnologias para efetivar sua utilização. O uso de ambientes virtualizados é determinante, entretanto, em muitos casos, estas tecnologias são escolhidas sem levar em conta o desempenho ou outros atributos não funcionais, tais como a garantia de qualidade de serviço (QoS), resiliência, confiabilidade, tolerância a falhas e escalabilidade, para citar algumas. O objetivo deste trabalho foca nas considerações principais para concepção de uma ferramenta completa de monitoramento e inspeção de sistemas virtualizados. A ideia é poder estimar as melhores configurações de software e hardware para plataformas virtualizadas sem que ocorra degradação de desempenho. Para demonstrar a efetividade da técnica foi implementada como exemplo uma ferramenta de propósito específico de monitoramento chamada VM-MON, descrita no presente trabalho

The rhizoferrin biosynthetic gene in the fungal pathogen Rhizopus delemar is a novel member of the NIS gene family

This work was supported by the Natural Sciences and Engineering Research Council of Canada award to MM (grant number 611181). C. Carroll thanks Simon Fraser University for a travel and research award.Iron is essential for growth and in low iron environments such as serum many bacteria and fungi secrete ferric iron-chelating molecules called siderophores. All fungi produce hydroxamate siderophores with the exception of Mucorales fungi, which secrete rhizoferrin, a polycarboxylate siderophore. Here we investigated the biosynthesis of rhizoferrin by the opportunistic human pathogen, Rhizopus delemar. We searched the genome of R. delemar 99–880 for a homologue of the bacterial NRPS-independent siderophore (NIS) protein, SfnaD that is involved in biosynthesis of staphyloferrin A in Staphylococcus aureus. A protein was identified in R. delemar with 22% identity and 37% similarity with SfnaD, containing an N-terminal IucA/IucC family domain, and a C-terminal conserved ferric iron reductase FhuF-like transporter domain. Expression of the putative fungal rhizoferrin synthetase (rfs) gene was repressed by iron. The rfs gene was cloned and expressed in E.coli and siderophore biosynthesis from citrate and diaminobutane was confirmed using high resolution LC–MS. Substrate specificity was investigated showing that Rfs produced AMP when oxaloacetic acid, tricarballylic acid, ornithine, hydroxylamine, diaminopentane and diaminopropane were employed as substrates. Based on the production of AMP and the presence of a mono-substituted rhizoferrin, we suggest that Rfs is a member of the superfamily of adenylating enzymes. We used site-directed mutagenesis to mutate selected conserved residues predicted to be in the Rfs active site. These studies revealed that H484 is essential for Rfs activity and L544 may play a role in amine recognition by the enzyme. This study on Rfs is the first characterization of a fungal NIS enzyme. Future work will determine if rhizoferrin biosynthesis is required for virulence in Mucorales fungi.PostprintPeer reviewe