Crystal structure of human nicotinic acid phosphoribosyltransferase

Nicotinic acid phosphoribosyltransferase (EC 2.4.2.11) (NaPRTase) is the rate-limiting enzyme in the three-step Preiss-Handler pathway for the biosynthesis of NAD. The enzyme catalyzes the conversion of nicotinic acid (Na) and 5-phosphoribosyl-1-pyrophosphate (PRPP) to nicotinic acid mononucleotide (NaMN) and pyrophosphate (PPi). Several studies have underlined the importance of NaPRTase for NAD homeostasis in mammals, but no crystallographic data are available for this enzyme from higher eukaryotes. Here, we report the crystal structure of human NaPRTase that was solved by molecular replacement at a resolution of 2.9. Å in its ligand-free form. Our structural data allow the assignment of human NaPRTase to the type II phosphoribosyltransferase subfamily and reveal that the enzyme consists of two domains and functions as a dimer with the active site located at the interface of the monomers. The substrate-binding mode was analyzed by molecular docking simulation and provides hints into the catalytic mechanism. Moreover, structural comparison of human NaPRTase with the other two human type II phosphoribosyltransferases involved in NAD biosynthesis, quinolinate phosphoribosyltransferase and nicotinamide phosphoribosyltransferase, reveals that while the three enzymes share a conserved overall structure, a few distinctive structural traits can be identified. In particular, we show that NaPRTase lacks a tunnel that, in nicotinamide phosphoribosiltransferase, represents the binding site of its potent and selective inhibitor FK866, currently used in clinical trials as an antitumoral agent

SEVA 3.0: An update of the Standard European Vector Architecture for enabling portability of genetic constructs among diverse bacterial hosts

© The Author(s) 2019.The Standard European Vector Architecture 3.0 database (SEVA-DB 3.0, http://seva.cnb.csic.es) is the update of the platform launched in 2013 both as a web-based resource and as a material repository of formatted genetic tools (mostly plasmids) for analysis, construction and deployment of complex bacterial phenotypes. The period between the first version of SEVA-DB and the present time has witnessed several technical, computational and conceptual advances in genetic/genomic engineering of prokaryotes that have enabled upgrading of the utilities of the updated database. Novelties include not only a more user-friendly web interface and many more plasmid vectors, but also new links of the plasmids to advanced bioinformatic tools. These provide an intuitive visualization of the constructs at stake and a range of virtual manipulations of DNA segments that were not possible before. Finally, the list of canonical SEVA plasmids is available in machine-readable SBOL (Synthetic Biology Open Language) format. This ensures interoperability with other platforms and affords simulations of their behaviour under different in vivo conditions. We argue that the SEVA-DB will remain a useful resource for extending Synthetic Biology approaches towards non-standard bacterial species as well as genetically programming new prokaryotic chassis for a suite of fundamental and biotechnological endeavours.The SEVA repository has been developed with funds of the SETH Project of the Spanish Ministry of Science [RTI 2018-095584-B-C42, MADONNA (H2020-FETOPEN-RIA-2017-1-766975), BioRoboost (H2020-NMBPBIO-CSA-2018), SYNBIO4FLAV (H2020-NMBP/0500) and LIAR (H2020-EU.1.2.1-686585)]; Contracts of the European Union and the S2017/BMD-3691 InGEMICS-CM funded by the Comunidad de Madrid (European Structural and Investment Funds) and the UK Engineering and Physical Science Research Council [EP/R019002/1]. Work at Raytheon was supported by the Air Force Research Laboratory (AFRL) and DARPA under contract FA875017CO184. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the AFRL and DARPA.This document does not contain technology or technical data controlled under either U.S. International Traffic in Arms Regulation or U.S. Export Administration Regulations. Funding for open access charge: H2020-NMBP-BIO-CSA-2018