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

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

A rotary mechanism for allostery in bacterial hybrid malic enzymes

This project was funded by BBSRC studentship 1500753 to C.J.H. and a BBSRC David Phillips fellowship to P.J.M. (BB/S010122/1).Bacterial hybrid malic enzymes (MaeB grouping, multidomain) catalyse the transformation of malate to pyruvate, and are a major contributor to cellular reducing power and carbon flux. Distinct from other malic enzyme subtypes, the hybrid enzymes are regulated by acetyl-CoA, a molecular indicator of the metabolic state of the cell. Here we solve the structure of a MaeB protein, which reveals hybrid enzymes use the appended phosphotransacetylase (PTA) domain to form a hexameric sensor that communicates acetyl-CoA occupancy to the malic enzyme active site, 60 Å away. We demonstrate that allostery is governed by a large-scale rearrangement that rotates the catalytic subunits 70° between the two states, identifying MaeB as a new model enzyme for the study of ligand-induced conformational change. Our work provides the mechanistic basis for metabolic control of hybrid malic enzymes, and identifies inhibition-insensitive variants that may find utility in synthetic biology.Publisher PDFPeer reviewe

Diffuse Light in the Virgo Cluster

We present deep optical imaging of the inner 1.5 x 1.5 degrees of the Virgo cluster to search for diffuse intracluster light (ICL). Our image reaches a 1 sigma depth of mu_v=28.5 mag/arcsec^2 -- 1.5 mag/arcsec^2 deeper than previous surveys -- and reveals an intricate web of diffuse intracluster light. We see several long (>100 kpc) tidal streamers, as well as a myriad of smaller-scale tidal tails and bridges between galaxies. The diffuse halo of M87 is traced out to nearly 200 kpc, appearing very irregular on these scales, while significant diffuse light is also detected around the M84/M86 pair. Several galaxies in the core are embedded in common envelopes, suggesting they are true physical subgroups. The complex substructure of Virgo's diffuse ICL reflects the hierarchical nature of cluster assembly, rather than being the product of smooth accretion around a central galaxy.Comment: 4 pages, 3 figures, accepted for publication in ApJ Letter

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

The Burrell-Optical-Kepler-Survey (BOKS). I. Survey Description and Initial Results

We present the initial results of a 40 night contiguous ground-based campaign of time series photometric observations of a 1.39 deg^2 field located within the NASA Kepler Mission field of view. The goal of this pre-launch survey was to search for transiting extrasolar planets and to provide independent variability information of stellar sources. We have gathered a data set containing light curves of 54,687 stars from which we have created a statistical sub-sample of 13,786 stars between 14 < r < 18.5 and have statistically examined each light curve to test for variability. We present a summary of our preliminary photometric findings including the overall level and content of stellar variability in this portion of the Kepler field and give some examples of unusual variable stars found within. We present a preliminary catalog of 2,457 candidate variable stars, of which 776 show signs of periodicity. We also present three potential exoplanet candidates, all of which should be observable by the Kepler mission

Deep CCD Surface Photometry of Galaxy Clusters I: Methods and Initial Studies of Intracluster Starlight

We report the initial results of a deep imaging survey of galaxy clusters. The primary goals of this survey are to quantify the amount of intracluster light as a function of cluster properties, and to quantify the frequency of tidal debris. We outline the techniques needed to perform such a survey, and we report findings for the first two galaxy clusters in the survey: Abell 1413, and MKW 7 . These clusters vary greatly in richness and structure. We show that our surface photometry reliably reaches to a surface brightness of \mu_v = 26.5 mags per arcsec. We find that both clusters show clear excesses over a best-fitting r^{1/4} profile: this was expected for Abell 1413, but not for MKW 7. Both clusters also show evidence of tidal debris in the form of plumes and arc-like structures, but no long tidal arcs were detected. We also find that the central cD galaxy in Abell 1413 is flattened at large radii, with an ellipticity of $\approx 0.8$, the largest measured ellipticity of any cD galaxy to date.Comment: 58 pages, 24 figures, accepted for publication in the Astrophysical Journal. Version has extremely low resolution figures to comply with 650k limit. High resolution version is available at http://burro.astr.cwru.edu/johnf/icl1.ps.gz Obtaining high resolution version is strongly reccomende