skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Crystallization and preliminary X-ray analysis of the O-methyltransferase NovP from the novobiocin-biosynthetic cluster of Streptomyces spheroides

Abstract

Monoclinic crystals of NovP, an O-methyltransferase from S. spheroides, were obtained and native X-ray data to 1.4 Å resolution were recorded. Crystals of recombinant NovP (subunit MW = 29 967 Da; 262 amino acids), an S-adenosyl-l-methionine-dependent O-methyltransferase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallized in space group P2, with unit-cell parameters a = 51.81, b = 46.04, c = 61.22 Å, β = 104.97°. Native data to a maximum resolution of 1.4 Å were collected from a single crystal at the synchrotron. NovP is involved in the biosynthesis of the aminocoumarin antibiotic novobiocin that targets the essential bacterial enzyme DNA gyrase.

Authors:
 [1]; ;  [2];  [1]
  1. Department of Biological Chemistry, John Innes Centre, Norwich NR4 7UH (United Kingdom)
  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 (United States)
Publication Date:
OSTI Identifier:
22360290
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 63; Journal Issue: Pt 3; Other Information: PMCID: PMC2330194; PMID: 17329822; PUBLISHER-ID: pu5181; OAI: oai:pubmedcentral.nih.gov:2330194; Copyright (c) International Union of Crystallography 2007; This is an open-access article distributed under the terms described at http://journals.iucr.org/services/termsofuse.html.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTALLIZATION; DIFFUSION; DNA; MONOCRYSTALS; RESOLUTION; SPACE GROUPS; SYNCHROTRONS

Citation Formats

Stevenson, Clare E. M., Freel Meyers, Caren L., Walsh, Christopher T., and Lawson, David M., E-mail: david.lawson@bbsrc.ac.uk. Crystallization and preliminary X-ray analysis of the O-methyltransferase NovP from the novobiocin-biosynthetic cluster of Streptomyces spheroides. United Kingdom: N. p., 2007. Web. doi:10.1107/S1744309107008287.
Stevenson, Clare E. M., Freel Meyers, Caren L., Walsh, Christopher T., & Lawson, David M., E-mail: david.lawson@bbsrc.ac.uk. Crystallization and preliminary X-ray analysis of the O-methyltransferase NovP from the novobiocin-biosynthetic cluster of Streptomyces spheroides. United Kingdom. doi:10.1107/S1744309107008287.
Stevenson, Clare E. M., Freel Meyers, Caren L., Walsh, Christopher T., and Lawson, David M., E-mail: david.lawson@bbsrc.ac.uk. Thu . "Crystallization and preliminary X-ray analysis of the O-methyltransferase NovP from the novobiocin-biosynthetic cluster of Streptomyces spheroides". United Kingdom. doi:10.1107/S1744309107008287.
@article{osti_22360290,
title = {Crystallization and preliminary X-ray analysis of the O-methyltransferase NovP from the novobiocin-biosynthetic cluster of Streptomyces spheroides},
author = {Stevenson, Clare E. M. and Freel Meyers, Caren L. and Walsh, Christopher T. and Lawson, David M., E-mail: david.lawson@bbsrc.ac.uk},
abstractNote = {Monoclinic crystals of NovP, an O-methyltransferase from S. spheroides, were obtained and native X-ray data to 1.4 Å resolution were recorded. Crystals of recombinant NovP (subunit MW = 29 967 Da; 262 amino acids), an S-adenosyl-l-methionine-dependent O-methyltransferase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallized in space group P2, with unit-cell parameters a = 51.81, b = 46.04, c = 61.22 Å, β = 104.97°. Native data to a maximum resolution of 1.4 Å were collected from a single crystal at the synchrotron. NovP is involved in the biosynthesis of the aminocoumarin antibiotic novobiocin that targets the essential bacterial enzyme DNA gyrase.},
doi = {10.1107/S1744309107008287},
journal = {Acta Crystallographica. Section F},
number = Pt 3,
volume = 63,
place = {United Kingdom},
year = {Thu Mar 01 00:00:00 EST 2007},
month = {Thu Mar 01 00:00:00 EST 2007}
}
  • An aromatic prenyltransferase (CloQ) from S. roseochromogenes that is implicated in clorobiocin biosynthesis has been crystallized in space group I4{sub 1}22. X-ray data to 2.2 Å resolution were collected in-house. Crystals of recombinant CloQ (subunit MW = 35 626 Da; 324 amino acids), an aromatic prenyltransferase from Streptomyces roseochromogenes, were grown by vapour diffusion. The protein crystallizes in space group I4{sub 1}22, with unit-cell parameters a = b = 135.19, c = 98.13 Å. Native data from a single crystal were recorded to a resolution of 2.2 Å in-house. Preliminary analysis of these data indicated that the asymmetric unit correspondsmore » to a monomer, giving an estimated solvent content of 60.6%. CloQ is involved in the biosynthesis of the aminocoumarin antibiotic clorobiocin, which targets the essential bacterial enzyme DNA gyrase.« less
  • Crystals of the type I Baeyer–Villiger monooxygenase (BVMO) MtmOIV from the biosynthetic pathway of mithramycin were obtained; the crystals diffracted to 2.69 Å resolution and belong to the monoclinic space group C2 (a = 143.5, b = 114.2, c = 137.8 Å β = 102.5°). Light scattering indicates that MtmOIV is a dimer of 127 kDa in solution, while in the crystalline state the data are consistent with two dimers in the asymmetric unit. The Baeyer–Villiger monooxygenase MtmOIV from Streptomyces argillaceus is a 56 kDa FAD-dependent and NADPH-dependent enzyme that is responsible for the key frame-modifying step in the biosynthesismore » of the natural product mithramycin. Crystals of MtmOIV were flash-cooled and diffracted to 2.69 Å resolution using synchrotron radiation on beamline SER-CAT 22-ID at the Advanced Photon Source. Crystals of MtmOIV are monoclinic and light-scattering data reveal that the enzyme forms dimers in solution. The rotation function suggests the presence of two dimers in the asymmetric unit. l-Selenomethionine-incorporated MtmOIV has been obtained. Structural solution combining molecular-replacement phases and anomalous phases from selenium is in progress.« less
  • Alditol oxidase oxidizes a range of alditols into the corresponding aldoses and is an interesting candidate for biotechnological applications. Crystals of alditol oxidase from S. coelicolor A3(2) were obtained by the hanging-drop vapour-diffusion method and diffracted to 1.1 Å resolution. Alditol oxidase is a 45 kDa enzyme containing a covalently bound FAD cofactor. This oxidase efficiently oxidizes a range of alditols to the corresponding aldoses. Owing to its substrate range and regioselectivity, this enzyme is an interesting candidate for biotechnological applications. Crystals of alditol oxidase from Streptomyces coelicolor A3(2) were obtained by the hanging-drop vapour-diffusion method and diffracted to 1.1more » Å resolution. The crystals belong to space group C2, with unit-cell parameters a = 107, b = 68, c = 58 Å, β = 94°. Crystals of seleno-l-methionine-labelled alditol oxidase were obtained after seeding the crystallization drops with native microcrystals and showed a diffraction limit of 2.4 Å.« less
  • To clarify the structural basis of sugar binding by BxlE at the atomic level, recombinant BxlE was crystallized using the hanging-drop vapour-diffusion method at 290 K. Together with the integral membrane proteins BxlF and BxlG, BxlE isolated from Streptomyces thermoviolaceus OPC-520 forms an ATP-binding cassette (ABC) transport system that mediates the uptake of xylan. To clarify the structural basis of sugar binding by BxlE at the atomic level, recombinant BxlE was crystallized using the hanging-drop vapour-diffusion method at 290 K. The crystals belonged to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 44.63, b = 63.27, cmore » = 66.40 Å, β = 103.05°, and contained one 48 kDa molecule per asymmetric unit (V{sub M} = 1.96 Å{sup 3} Da{sup −1}). Diffraction data collected to a resolution of 1.65 Å using a rotating-anode X-ray source gave a data set with an overall R{sub merge} of 2.6% and a completeness of 91.3%. A data set from a platinum derivative is being used for phasing by the SAD method.« less