Purification, crystallization and preliminary crystallographic analysis of DehI, a group I alpha-haloacid dehalogenase from Pseudomonas putida strain PP3.
Pseudomonas putida strain PP3 produces two dehalogenases, DehI and DehII, which belong to the group I and II alpha-haloacid dehalogenases, respectively. Group I dehalogenases catalyse the removal of halides from D-haloalkanoic acids and in some cases also the L-enantiomers, both substituted at their chiral centres. Studies of members of this group have resulted in the proposal of general catalytic mechanisms, although no structural information is available in order to better characterize their function. This work presents the initial stages of the structural investigation of the group I alpha-haloacid dehalogenase DehI. The DehI gene was cloned into a pET15b vector with an N-terminal His tag and expressed in Escherichia coli Nova Blue strain. Purified protein was crystallized in 25% PEG 3350, 0.4 M lithium sulfate and 0.1 M bis-tris buffer pH 6.0. The crystals were primitive monoclinic (space group P2(1)), with unit-cell parameters a = 68.32, b = 111.86, c = 75.13 A, alpha = 90, beta = 93.7, gamma = 90 degrees , and a complete native data set was collected. Molecular replacement is not an option for structure determination, so further experimental phasing methods will be necessary.
Schmidberger
Jason W
JW
School of Pharmacology and Medicine, University of Western Australia, Crawley, Western Australia, Australia.
Wilce
Jackie A
JA
Weightman
Andrew J
AJ
Wilce
Matthew C J
MC
D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D006867:Hydrolases / Q000737:chemistry* / Q000302:isolation & purification; D016958:Pseudomonas putida / Q000201:enzymology*; D013237:Stereoisomerism
D001426:Bacterial Proteins; D006867:Hydrolases; C034507:2-haloacid dehalogenase