Expression, purification, crystallization and preliminary X-ray crystallographic studies of the trehalulose synthase MutB from Pseudomonas mesoacidophila MX-45.
The trehalulose synthase (MutB) from Pseudomonas mesoacidophila MX-45, belonging to glycoside hydrolase family 13, catalyses the isomerization of sucrose to trehalulose (alpha-D-glucosylpyranosyl-1,1-D-fructofuranose) and isomaltulose (alpha-D-glucosylpyranosyl-1,6-D-fructofuranose) as main products and glucose and fructose in residual amounts from the hydrolytic reaction. To date, a three-dimensional structure of a sucrose isomerase that produces mainly trehalulose, as is the case for MutB, has been lacking. Crystallographic studies of this 64 kDa enzyme have therefore been initiated in order to contribute to the understanding of the molecular basis of sucrose decomposition, isomerization and of the selectivity of this enzyme that leads to the formation of different products. The MutB protein has been overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. Two different crystal forms have been obtained: one diffracts X-rays to 1.6 A resolution using synchrotron radiation and belongs to space group P1, with unit-cell parameters a = 63.8, b = 72.0, c = 82.2 A, alpha = 67.5, beta = 73.1, gamma = 70.8 degrees, while the other form diffracts to 1.8 A resolution using synchrotron radiation and belongs to space group P2(1), with unit-cell parameters a = 63.7, b = 85.9, c = 119.7 A, beta = 97.7 degrees. A molecular-replacement solution has been found using the structure of the isomaltulose synthase (PalI) from Klebsiella sp. LX3 as a search model.
Ravaud
Stéphanie
S
Laboratoire de BioCristallographie, Institut de Biologie et Chimie des Protéines, CNRS et Université Claude Bernard Lyon 1, UMR 5086, IFR 128 BioSciences Lyon-Gerland, F-69367 Lyon CEDEX 07, France.
Watzlawick
Hildegard
H
Haser
Richard
R
Mattes
Ralf
R
Aghajari
Nushin
N
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D003460:Crystallization; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C099692:trehalose synthase; D019751:Intramolecular Transferases; C042002:isomaltulose synthase