Molecular replacement in the 'twilight zone': structure determination of the non-haem iron oxygenase NovR from Streptomyces spheroides through repeated density modification of a poor molecular-replacement solution.
Crystals of recombinant NovR (subunit MW = 29 924 Da; 270 amino acids), a non-haem iron oxygenase from Streptomyces spheroides, were grown by vapour diffusion. The protein crystallized in space group C2, with unit-cell parameters a = 86.69, b = 139.38, c = 100.82 A, beta = 101.18 degrees . Native data were collected to a resolution of 2.1 A from a single crystal at a synchrotron and a molecular-replacement solution was obtained using the program AMoRe. The starting phase information was very poor and did not permit model building. Phases were subsequently improved using a combination of fourfold averaging and very gradual phase extension in the program DM to yield an interpretable map. NovR belongs to a novel class of non-haem iron oxygenases that share sequence similarity with class II aldolases. It is predicted to perform two consecutive oxidative decarboxylation steps in the biosynthesis of the prenylated hydroxybenzoic acid moiety of the aminocoumarin antibiotic novobiocin.
Keller
Sascha
S
Department of Biological Chemistry, John Innes Centre, Norwich NR4 7UH, England.
Pojer
Florence
F
Heide
Lutz
L
Lawson
David M
DM
D003460:Crystallization; D018360:Crystallography, X-Ray; D006418:Heme / Q000737:chemistry*; D008958:Models, Molecular; D010105:Oxygenases / Q000737:chemistry*; D017433:Protein Structure, Secondary; D012996:Solutions; D013302:Streptomyces / Q000201:enzymology*
D012996:Solutions; D006418:Heme; D010105:Oxygenases; C433124:iron(II) oxygenase