PROTEINS: Structure, Function, and Genetics 24514-515 (1996) CRYSTALLIZATION ANNOUNCEMENTS Crystallization and Preliminary X-Ray Crystallographic Studies of Ketosteroid Isomerase From Pseudomonas putida Biotype B Byung-HaOh,' Suhng Wook Kim,' Seong-EonRyu: Sang-So0Kim: Mi-Kyung Yoon: and Kwan Yong Choi' 'Department of Life Science, Pohang University of Science and Technology, Pohang, Kyungbuk, 790-784,Korea (S.); 2Protein Engineering Research Group, Korean Research Institute for Bioscience and Biotechnology, KIST, Daejon, 305-333, Korea (S.); 3R&D Center, Lucky Ltd., Daejon, 305343, Korea (S.) ABSTRACT The A5-3-ketosteroidisomerase from Pseudomonas putida biotype B has been crystallized. The crystals belong to the space group P2,2,2, with unit cell dimensions of a = 36.48 A, b = 74.30 A, c =96.02 A, and contain one homodimer per asymmetric unit. Native diffraction data to 2.19 A resolution have been obtained from one crystal at room temperature indicating that the crystals are quite suitable for structure determination by multiple isomorphous replacement. o ISXI Wiley-Liss, Inc. Key words: ketosteroid isomerase, crystallization, protein-steroid interaction Many isomerases use proton transfer between the catalytic residues of the enzyme and the substrate in order to interconvert isomers.1,2 The A5-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of a variety of A5-3-ketosteroids to A4-3-ketosteroids by a stereospecificintramolecular transfer of the 4P-proton to the 6P p ~ s i t i o n KSI . ~ from Comamonas testosteroni (renamed from Pseudomonas testosteroni4),which can live on steroids as a sole carbon source,has been an object of continuous investigation for many years5p6 as a prototype for understanding the isomerization mechanism and the protein:steroid interactions. The enzyme exists as a highly stable homodimer in ~ o l u t i o nIt. ~is a very efficient enzyme showing the second-order rate constant KcatlKm to be 3.0 x 10' (s-'M-') for 5-androstene-3,17-dione, the most widely employed substrate.8 Site-directed mutagenesis have strongly suggested residue Tyrl4 as the general acid protonating the 3-carbonyl oxygen of the steroid substrates, and residue Asp38 as a general base abstracting the 4pproton of the substrates during the formation of dienolate intermediate. X-ray crystallographic studies on C. testosteroni KSI started in 1976 with crystals of the enzyme that had a n unusually large size for one 0 1996 WILEY-LISS, INC. unit cell axis (a=b = 65.4& c = 504 presenting technical difficulties in collectinghigh resolution diffraction data. The multiple isomorphous replacement work with this crystal form led t o an initial interpretation of the electron density map at 6 A resolution in 1984.14 Subsequently, a 2.5 A resolution native data set was collected using synchrotron radiation. However, the 6 structure resisted refinement against the 2.5 A resolution data with unacceptably high crystallographic R-factor (personal communication). Until now, the coordinates of KSI from C. testosteroni or from any other organism are not available in the Protein Data Bank. The lack of accurate high resolution crystal structures of KSI and KS1:steroid complexes has been an obstacle in understanding the fine details of the enzyme mechanism, and in obtaining information on the protein: steroid interactions a t the atomic level. Recently, the entire gene encoding a homologous KSI from P . putida biotype B was cloned and overexpressed in E. coZi.15 The polypeptide of the KSI from P . putida biotype B is six residues longer than that of C . testosteroni KSI (125 residues). When amino acid sequences of the two KSI are aligned, 44 amino acids match identically. 15,16 Among those, the active site residues of the C. testosteroni KSI, Asp38 and Tyrl4, are conserved as Asp40 and Tyrl6 in P . putida KSI. When the Asp40 and Tyrl6 of the P. putida KSI were replaced with asparagine and phenylalanine, respectively, both purified mutant enzymes exhibited a profound decrease in the catalytic activity and in the K m value as were observed in the similar mutant enzymes of the C. testosteroni KSI.17 Thus, the catalytic mechanisms of the two KSIs appear to be very similar. a Received October 26, 1995; accepted October 27, 1995. Address reprint requests to Byung-Ha Oh, Department of Life Science, Pohang University of Science and Technology, Pohang, Kyungbuk, 790-784, Korea (S.) 515 P. PUTIDA KETOSTEROID ISOMERASE Since we were able to obtain a large amount of highly purified P. putidu KSI by employing a n affinity column chromatography,15 we initiated crystallization work in a hope to obtain crystals of the enzyme that are suitable for high resolution structure determination. Initial crystallization conditions were discovered using the screening method of sparse matrix sampling,” and subsequently the condition was modified to obtain fewer and larger crystals in a droplet. Drops of 2 p1 of protein solution (20 mg/ml, in a buffer solution containing 20 mM p-mercaptoethanol and 50 mM TrisCl, pH 7.0) were mixed in equal volume with the precipitant containing 0.1 M sodium acetate, 0.05 M sodium cacodylate (pH 6.5), and 23% polyethyleneglycol 400. The mixture on a cover slip was then equilibrated against 1 ml of the precipitant in 24-well tissue culture plates at 22°C. In less than 2 days, many small crystals appeared in the droplets. It was quite difficult to suppress too many nucleations, but after many trials it was found that mixing the protein sample and the precipitant in a 2:5 volume ratio proved most useful for obtaining crystals of about 0.1 x 0.1 x 0.2 mm in size. Though relatively small, the crystals diffracted very well. With one such KSI crystal in a droplet, diffraction data to 2.19 A resolution (94.7% = 4.6%) were obtained on a Rigaku complete, R, R-AXIS IIc imaging plate system (Rigaku, Tokyo, Japan) operated at 40 kV and 120 mA using CuKa radiation at room temperature. Using an auto indexing program provided with the Rigaku data reduction software, and examining the diffraction data set, we found that the crystals belong to the orthorhombic space group P2,2,2, with unit cell dimensions of a=36.48 A, b=74.30 A, c=96.02 A. Given the molecular weight of KSI (14,536 Da), we calculated that the crystal volume per unit molecular weight (V,) is 2.24 A3Da with a solvent content of 45.1% by volume1s when one unit cell was assumed to contain eight protomers. Thus, the asymmetric unit of the crystals is very likely to contain one homodimer of KSI. The favorable characteristics of P. putidu KSI crystals, as described above, provides a new opportunity to determine the crystal structure of KSI at high resolution. In addition, P. putidu KSI contains three cysteine residues (whereas C. testosteroni KSI contains none) that are useful in obtaining isomorphous crystals derivatized by mercury-based heavy atom compounds. The structure determination of P. putidu KSI by the multiple isomorphous replacement method is in progress. ACKNOWLEDGMENTS This work was supported by The Basic Science Research Fund of Pohang University of Science and Technology, and in part by the Non-Directed Research Fund, Korean Research Foundation, and by the Research Center for New Bio-Materials in Agriculture. REFERENCES 1. Fersht, A.R. “Enzyme Structure and Mechanism.” 2nd edit. New York Freeman, 1984. 2. Walsh, C. “Enzymatic Reaction Mechanisms.” New York: Freeman, 1984. 3. Batzold, F.H., Benson, A.M., Covey, D.F., Robinson, C.H., Talalay, P. The A5-3-ketosteroid isomerase reaction: Catalytic mechanism, specificity, and inhibition. Adv. Enzyme Regul. 14:243-267, 1976. 4. Tamaoka, J., Ha, D.-M., Komagata, K. 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