Site-directed mutagenesis to enable and improve crystallizability of Candida tropicalis (3R)-hydroxyacyl-CoA dehydrogenase Mari S. Ylianttila a , Yong-Mei Qin b , J. Kalervo Hiltunen a , Tuomo Glumoff a, * a Biocenter Oulu and Department of Biochemistry, University of Oulu, P.O. Box 3000, FIN-90014 University of Oulu, Finland b National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, PR China Received 17 August 2004 Available online 21 September 2004 Abstract The N-terminal part of Candida tropicalis MFE-2 (MFE-2(h2D)) having two (3R)-hydroxyacyl-CoA dehydrogenases with differ- ent 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 a = b = c = 90° 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 2.2 A ˚ at a conventional X-ray source. Ó 2004 Elsevier Inc. All rights reserved. Keywords: 17b-HSD; MFE-2; b-Oxidation; Peroxisome; SDR Fatty acid degradation in living organisms occurs mainly via b-oxidation, which is a spiral pathway con- sisting of four enzymatic steps. Following oxidation of fatty acyl-CoA esters at the b-carbon, the spiral contin- ues by addition of a hydroxy group to the b-carbon; this hydroxyl is further oxidized in the third step. The multi- functional enzymes (MFEs) catalyzing these steps con- tain the latter two activities, 2-enoyl-CoA hydratase (‘‘hydratase’’) and hydroxyacyl-CoA dehydrogenase (‘‘dehydrogenase’’). MFEs can also have additionally either D 3 -D 2 -enoyl-CoA isomerase or 3-ketoacyl-CoA thiolase activities, depending on species and cellular ori- gin of the enzyme. Each cycle of the spiral shortens the CoA-activated fatty acid by two carbons, since the last step of b-oxidation involves a thiolytic cleavage at the b-carbon and re-entering of the remaining fatty acyl body into the cycle. Two types of multifunctional enzymes exist in mam- malian peroxisomes: MFE type 1 and 2. They are not sequence related. Based on kinetic in vitro properties of the enzyme, MFE-1 was originally thought to be responsible for the b-oxidation of straight chain fatty acids [1–3]. However, MFE-1 null mutant mice are both clinically and biochemically healthy, leaving the func- tion of MFE-1 under in vivo conditions an enigma [4,5]. In contrast to MFE-1, a number of MFE-2 defi- ciencies have been identified in peroxisomal disease patients suffering from psychomotor dysfunction, hypo- tonia, and craniofacial dysmorphosis. These patients show accumulation of both very long chain and a-meth- yl branched chain fatty acids as well as C27 intermedi- ates of bile acid synthesis, demonstrating a key role of MFE-2 in the degradation of these metabolites in mam- malian peroxisomes [6–8]. A striking difference between MFE-1 and MFE-2 is that the two-enzyme step from trans-2-enoyl-CoA to 3-keto-metabolites uses opposite stereochemistry with respect to the (3R)-hydroxyacyl- CoA intermediate [9,10]. Because the peroxisomal 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.09.013 * Corresponding author. Fax: +358 8 553 1141. E-mail address: tuomo.glumoff@oulu.fi (T. Glumoff). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 324 (2004) 25–30 BBRC