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

Kinetic landscape of a peptide-bond-forming prolyl oligopeptidase

We thank Dr. Rafael Guimaraes da Silva for helpful discussions on enzyme kinetics. We also thank Professor David Lilley, Dr. Alasdair Freeman and Dr. Anne-Cecile Declais at the University of Dundee for training and usage of their QFM-4000 quenched-flow apparatus.Prolyl oligopeptidase B from Galerina marginata (GmPOPB) has recently been discovered as a peptidase capable of breaking and forming peptide bonds to yield a cyclic peptide. Despite the relevance of prolyl oligopeptidases in human biology and disease, a kinetic analysis pinpointing rate-limiting steps for a member of this enzyme family is not available. Macrocyclase enzymes are currently exploited to produce cyclic peptides with potential therapeutic applications. Cyclic peptides are promising drug-like molecules due to their stability and conformational rigidity. Here we describe an in-depth kinetic characterization of a prolyl oligopeptidase acting as a macrocyclase enzyme. By combining steady-state and pre-steady-state kinetics, we put forward a kinetic sequence in which a step after macrocyclization limits steady-state turnover. Additionally, product release is ordered, where cyclic peptide departs first followed by the peptide tail. Dissociation of the peptide tail is slow and significantly contributes to the turnover rate. Furthermore, trapping of the enzyme by the peptide tail becomes significant beyond initial-rate conditions. The presence of a burst of product formation and a large viscosity effect further support the rate-limiting nature of a physical step occurring after macrocyclization. This is the first detailed description of the kinetic sequence of a macrocyclase enzyme from this class. GmPOPB is amongst the fastest macrocyclases described to date, and this work is a necessary step towards designing broad specificity efficient macrocyclases.Publisher PDFPeer reviewe

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

cyberaCTIve: a STIX-based Tool for Cyber Threat Intelligence in Complex Models

Cyber threat intelligence (CTI) is practical real-world information that is collected with the purpose of assessing threats in cyber-physical systems (CPS). A practical notation for sharing CTI is STIX. STIX offers facilities to create, visualise and share models; however, even a moderately simple project can be represented in STIX as a quite complex graph, suggesting to spread CTI across multiple simpler sub-projects. Our tool aims to enhance the STIX-based modelling task in contexts when such simplifications are infeasible. Examples can be the microgrid and, more in general, the smart grid.Comment: 11 pages, 8 figures, technical repor

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

Avaliação da presença de heterorresistência à polimixina B em Klebsiella pneumoniae produtora de Klebsiella pneumoniae carbapenemase (KPC) isolada em hemoculturas de paciente em tratamento com este antibiótico

Klebsiella spp. pertencente à família Enterobacteriaceae é um importante patógeno responsável por causar graves infecções comunitárias e nosocomiais. Atualmente, as taxas de resistência aos carbapenêmicos identificadas em isolados dessa espécie vem aumentando, gerando grande preocupação quanto ao manejo no tratamento, já que são poucas as opções eficazes de antibióticos no mercado. Desse modo, medicamentos antigos como as polimixinas retornaram à prática clínica, mas relatos de fenômenos de heterorresistência a esta classe de antibióticos já são descritos. O objetivo deste trabalho foi avaliar a presença do fenômeno de heterorresistência em amostras de Klebsiella pneumoniae isoladas de paciente hospitalizada em tratamento com polimixina B. A pesquisa da heterorresistência foi realizada através da análise do perfil populacional (PAP), que trata-se da observação de subpopulações resistentes quando inoculadas diferentes diluições do isolado em meio de cultura sólido contendo antibióticos em concentrações maiores que aquela correspondente à concentração inibitória mínima (CIM). Também foi realizada técnica de tipagem molecular (PFGE), entre as populações originais e respectivas subpopulações resistentes e ensaios de microdiluição em caldo para determinação das CIMs. Foram avaliados 7 isolados de Klebsiella pneumoniae (Kp1.1, Kp2.1, Kp2.2, Kp2.3, Kp3.1, Kp3.2 e Kp3.3), coletados de paciente hospitalizada que apresentava infecção em corrente sanguínea, em 3 diferentes dias. As suas CIMs para polimixina B, realizadas por microdiluição em caldo, foram entre 0,125 e 16μg/mL, demonstrando valor mais elevado gradativamente, até atingir completa resistência no último grupo de amostras coletadas. Todas as amostras com perfil sensível a polimixina B foram analisadas através do PAP e demonstraram heterorresistência, das quais três cresceram em concentrações acima do breakpoint para polimixinas (Kp1.1H em 6 μg/mL, Kp2.1H em 16 μg/mL, Kp2.2H em 8 μg/mL e Kp2.3H em 64 μg/mL) e uma cresceu em 1 μg/mL (Kp1.1H’). Suas CIMs após passagem em meio livre de antibiótico se mantiveram altas em sua grande maioria – entre 1 μg/mL, e 64μg/mL. Através de análise do PFGE verificou-se a presença de dois perfis clonais. A partir de série bioquímica e sequenciamento do gene 16S pode-se observar que na primeira amostra encontravam-se duas espécies, Klebsiella oxytoca e Klebsiella pneumoniae, porém, após exposição ao tratamento com polimixina B, a segunda espécie se manteve nas amostras subsequentes e a K. oxytoca desapareceu. As subpopulações heterorresistentes representaram 0,00001% a 0,0045% de suas populações originais

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

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

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

Tools for modelling and simulating the Smart Grid

The Smart Grid (SG) is a Cyber-Physical System (CPS) considered a critical infrastructure divided into cyber (software) and physical (hardware) counterparts that complement each other. It is responsible for timely power provision wrapped by Information and Communication Technologies (ICT) for handling bi-directional energy flows in electric power grids. Enacting control and performance over the massive infrastructure of the SG requires convenient analysis methods. Modelling and simulation (M&S) is a performance evaluation technique used to study virtually any system by testing designs and artificially creating 'what-if' scenarios for system reasoning and advanced analysis. M&S avoids stressing the actual physical infrastructure and systems in production by addressing the problem in a purely computational perspective. Present work compiles a non-exhaustive list of tools for M&S of interest when tackling SG capabilities. Our contribution is to delineate available options for modellers when considering power systems in combination with ICT. We also show the auxiliary tools and details of most relevant solutions pointing out major features and combinations over the years