Site-directed mutagenesis to enable and improve crystallizability of Candida tropicalis (3R)-hydroxyacyl-CoA dehydrogenase.
The N-terminal part of Candida tropicalis MFE-2 (MFE-2(h2Delta)) having two (3R)-hydroxyacyl-CoA dehydrogenases with different substrate specificities has been purified and crystallized as a recombinant protein. The expressed construct was modified so that a stabile, homogeneous protein could be obtained instead of an unstabile wild-type form with a large amount of cleavage products. Cubic crystals with unit cell parameters a=74.895, b=78.340, c=95.445, and alpha=beta=gamma=90 degrees were obtained by using PEG 4000 as a precipitant. The crystals exhibit the space group P2(1)2(1)2(1) and contain one molecule, consisting of two different (3R)-hydroxyacyl-CoA dehydrogenases, in the asymmetric unit. The crystals diffract to a resolution of 2.2A at a conventional X-ray source.
Ylianttila
Mari S
MS
Biocenter Oulu and Department of Biochemistry, University of Oulu, P.O. Box 3000, FIN-90014 University of Oulu, Finland.
Qin
Yong-Mei
YM
Hiltunen
J Kalervo
JK
Glumoff
Tuomo
T
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000737:chemistry* / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D041022:Candida tropicalis / Q000201:enzymology* / Q000235:genetics; D003460:Crystallization; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment
D005656:Fungal Proteins; D011994:Recombinant Proteins; D015094:3-Hydroxyacyl CoA Dehydrogenases