PROTEINS: Structure, Function, and Genetics 13:158-161 (1992)

Crystallization and Preliminary X-Ray Diffraction Studies of Two Mutants of Lactate Dehydrogenase From Bacillus stearothermophilus Kui Huang,' R. Kodandapani,' Helmut Kallwass: James K. Hogan: Wendy Parris: James D. Friesen: Marvin Gold: J. Bryan Jones: and Michael N.G. James' 'Medical Research Council of Canada Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7;'Department of Chemistry, University of Toronto, M 5 S 1 A l and 3Department of Medical Genetics, University of Toronto M 5 S 1A8, Toronto, Ontario, Canada Key words: bacterial lactate dehydrogenase, X-ray crystallography, site-directed mutation, stereospecificity, image plates

ABSTRACT Bacillus stearothermophilus lactate dehydrogenase, one of the most thermostable bacterial enzymes known, has had its three-dimensional structure solved, the gene coding for it has been cloned, and the protein can be readily overexpressed. Two mutants of the enzyme have been prepared. In one, Argl7l was changed to Trp (R171W) and Gln102 was changed to Arg (Q102R). In the other, the mutation Q102R was maintained, but Argl7l was changed to Tyr (R171Y). In addition, an inadvertent C97G mutant was present. Both mutants have been crystallized by the hanging drop vapor diffusion method at room temperature. Bipyrimidal crystals have been obtained against (NH,),SO, in 50 mM piperazine HCl buffer. The crystals belong to space group P6,22 (P6,22) (whereas the native enzyme, the structure of which has been solved by Piontek et al., Proteins 7:74-92, 1990) crystallized in the space group P6,) with a = 102.3 A, c = 168.6 A for the R171W, Q102R, C97G triple mutant, and a = 98.2 A; c = 162.1 A for the R171Y, Q102R, C97G mutant. These crystal forms appear to contain one-quarter of a tetramer (Mr 135,000) in the asymmetric unit and have V , values of 3.8 and 3.3 A3/dalton,respectively). The R171W mutant diffracts to 2.5 A and the R171 Y mutant to approximately 3.5 A. o 1992 Wiley-Liss, Inc. INTRODUCTION Lactate dehydrogenase (LDHase: E.C.1.1.1.27) is an NAD (nicotinamide adenine dinucleotide) dependent enzyme involved in the conversion of lactate to pyruvate in the final step of the anaerobic glycolytic pathway.' The active form of the enzyme is usually a tetramer with approximate molecular weight of 140,000 daltons; the tetramer has local 222 symmetry. Prominent isozymes of the enzyme in eukary0 1992 WILEY-LISS. INC.

otes are referred to as A or M (muscle) and B or H (heart). Many physical, chemical, immunological, and enzymatic studies have shown that the characteristics of the M and H forms from the same species are less similar than either the M forms of different species or their H forms are among themselves.2 Several high resolution structures of LDHase from various organisms including that of a bacterial LDHase and their ternary complexes are available. These include the structure of dogfish-M4-apolactate dehydrogenase? mouse testicular lactate deh y d r ~ g e n a s e , ~pig . ~ heart lactate dehydrogenase complexed with S-lac-NAD+,6 and Bacillus stearothermophilus L D H ~ s Procaryotic ~.~ LDHases are regulated by fructose 1,6-bisphosphate (FBP).' Residues His188 and Arg173 are conserved in bacterial LDHases and are in the vicinity of the FBP binding site.3 Bacillus stearothermophilus (BS) belongs to a group of microorganisms whose enzymes, in general, show markedly elevated thermostability compared t o those from the mesophilic specie^.^,'^ BSLDHase shows no loss of activity up to 80°C for 30 minutes. It is less efficient a t lower temperatures compared to the less thermostable LDHases." A biphasic Arrhenius plot indicates a possible conformational change around 45°C accompanied by concomitant changes in thermodynamic activation parameters.lO-" The structural property seems to explain their thermal stability. l2 BSLDHase is the most studied bacterial LDHase, which is activated by FBP.13 The gene14,15 and the amino acid16 sequences have been determined. Additional information is available about its quaternary structural and its kinetic and ligand binding p r ~ p e r t i e s . " , ~ The ~ , ~ ~gene coding for BSLDHase has been c10ned.l~The cloning of the gene and its overexpression have facilitated the site directed mutation of functionally important resi-

Received September 10, 1991; revision accepted October 25, 1991. Address reprint requests to Dr. Michael N.G. James, Department of Biochemistry, University of Alberta, 474 Medical Sciences Building, Edmonton, Alberta, Canada T6G 2H7.

CRYSTALLIZATION OF BSLDH MUTANTS

dues. 13.18-25 Site-directed mutagenesis has been used to alter the specificity of the enzyme from lactate to malate.19~21 In the present work, the crystallization and preliminary X-ray investigations of two triple mutants introducing changes a t residues 102 and 171 of the BSLDHase are described. Gln102 is in a region towards which the methyl group of pyruvate is oriented in the ES-complex, and graphics analyses of the molecular stereochemistry suggest that with large or branched substituents adjacent to the a-keto group, there are unfavorable interactions with this active site residue. In fact, it has already been shown that when Gln102 was replaced with the smaller Asn by site-directed mutagenesis in an attempt to expand the active site volume, the specificity towards branched substrates was broadened." Enzymes in which both Ql02 and R171 are altered simultaneously can be very useful in probing both the substrate and stereospecificity of LDH. Based on the best X-ray data for the active site, His195 and ArglO9 polarize the carbonyl group to facilitate hydride transfer from NADH to the carbony1 carbon. Argl7l forms a very tight electrostatic bond to the carboxylate of pyruvate and Gln102 and Thr246 restrict the space available to accommodate the side chain. Argl71 forms such a n exceptionally strong interaction with the substrate's carboxylate that it has been considered to be the residue contributing the major orienting force to make the reaction stereospecific. Replacement of Argl7 1by uncharged residues was therefore undertaken to probe these hypotheses. The mutants were prepared and purified by methods described p r e v i ~ u s l y Subsequently, .~~~~~ it was discovered that the starting Q102R contained an inadvertent C97G mutation. The X-ray studies are currently being performed on the triple mutants (R171W, Q102R, C97G) and (R171Y, Q102R, C97G).

EXPERIMENTAL RESULTS The two mutants were crystallized under similar conditions, by the hanging-drop, vapor-diffusion method a t room temperature. For the first triple mutant (R171W, Q102R, C97G), the protein concentration was 20 pglml and the well buffer used was 50 mM piperazine-HC1 a t pH 7.8. The precipitant was 38% (NH,),SO,. For the second triple mutant (R171Y, Q102R, C97G), all the conditions of crystallization were the same except that the precipitant concentration was 36% (NH,),SO, and the buffer had a pH of 7.0. Drops of protein solution (6 p1) were mixed with an equal volume of precipitant, placed on clean coverslips, and suspended over wells containing the precipitant. Crystals appeared in 5 days growing to full size (1.2 x 0.8 X 0.6 mm3) in 2 weeks. In both the cases only one crystal form was observed, which was a colorless bipyramid. Great

159

care had to be taken in mounting the crystals, as the crystals were very fragile and would otherwise develop cracks. Preliminary characterization of these crystals by precession photography was done on a Rigaku RU200 rotating anode X-ray generator operated a t 40 kV and 150 mA. The precession photographs of the hkO, h k l , Okl, and l k l levels indicated that the space group of both the triple mutant crystals could probably be P6,22/P6,22. It is interesting to note that the native enzyme crystallized in a different, nonisomorphic space group P6,,7 with the unit cell parameters a = b = 86.9 A, c = 357.3 A. The crystals of the R171W, QlOZR, C97G mutant have unit cell parameters of a = 102.3 A; c = 168.6 A; space group = P6,22 (P6,22). These crystals diffract to 2.5 A resolution. They cease to diffract well after having been exposed to X-rays for 12 hours. Assuming that there is one-quarter of a tetramer in the asymmetric unit (i.e., 114 of 135,000), one can calculate a V, equal to 3.8.26 This means that the molecule must lie on one of the 222 positions in the unit cell. The alternative is to assume that there are six tetramers in the unit cell, or one-half a tetramer per asymmetric unit. In this case the calculated V, would be 1.9 Aldalton. Although both of these cases are possible, the former seems more plausible. The reason being, if the V, were 1.9 then the crystal would be densely packed with little solvent content (35%); thus it should therefore diffract very well. But it has been found that the crystal does not diffract beyond 2.5 A and indeed diffracts only weakly at the higher angles, suggesting that there is probably much more solvent in the unit cell, following the V, = 3.8 (68% solvent). The crystals of the R171Y, Q102R, C97G mutant have unit cell parameters of a = 98.2 A; c = 162.1 A; space group = P6,22). These crystals do not diffract beyond 3.5 A and the quality of the diffraction pattern is not so good compared to the R171W, Q102R, C97G mutant patterns. Given the same arguments as in the previous case, of the two possible V, values of 3.3 and 1.67 A3/dalton, the former again seems more plausible. That means a volume equal to 63% of the cell volume is filled with solvent and there is a quarter of a molecule (tetramer) per asymetric unit, in one of the 222 symmetry positions of the unit cell. The above results for both mutants have been summarized in Table I. At present a 2.5 A resolution data set has been collected for the triple mutant R171W, QlOZR, C97G at the synchrotron source of the Photon Factory, Tsukuba, Japan, using screenless Weissenberg geometryz7with imaging plates." The digitized image plates have been processed by the program WEIS.29Initial merging and scaling of the data suggests that 90% of the possible data have been observed up to 2.5 A resolution. We intend to solve the structure by the molecular replacement method3'

160

K. HUANG ET AL.

TABLE I. Crystallographic Data for BSLDH Mutants

Space group Mutant I R171W, Q102R, C97G

Mutant I1 R171Y, Q102R, C97G

P6,22 (P6,22) P6,22 (P6,22)

Cell Parameters CA aA

Solvent volume

Diffract to

VM

102.3

168.6

3.8

68%

2.5A

98.2

162.1

3.3

63%

3.5A

using the coordinates of suitable LDHase structures that are already available.

ACKNOWLEDGMENTS This work was supported by the Medical Research Council of Canada in a grant to the MRC Group in Protein Structure and Function (Alberta) and by a Natural Sciences and Engineering Research Council of Canada Strategic Grant No. 45549 (Toronto). NOTE ADDED IN PROOF Recent molecular replacement calculations, using data collected at the Photon Factory, Tsukuba, Japan, indicate that the space group of the crystals of the mutant enzymes is P6, instead of P6,22/P6,22 as reported in Table I. This means that there are 2 subunits of the mutant BSLDH in the crystallographic asymmetric unit.

REFERENCES 1. Holbrook, J.J., Liljas, A., Steindel, S.J., Rosmann, M.G. Lactate dehydrogenase. In: “The Enzymes” (Boyer, P.D., ed.), 3rd ed. New York: Academic Press, 1975:191-292. 2. Adams, M.J., Ford, G.C., Koekoek, R., Lentz, P.J., Jr., McPherson, A,, Jr., Rossmann, M.G., Smiley, J.E., Schevitz, R.W., Wonacott, A.J. Structure of lactate dehydrogenase a t 2.8 A resolution. Nature (London) 227:1098-1103, 1970. 3. Abad-Zapatero, C., Griffith, J.P. Sussman, J.L., Rossmann, M.G. Refined crystal structure of dogfish M, apolactate dehydrogenase. J . Mol. Biol. 198:445-467, 1987. 4. Musick, W.D.L., Rossmann, M.G. The structure of mouse testicular lactate dehydrogenase isoenzyme C, a t 2.9 A resolution. J . Biol. Chem. 254:7611-7620, 1979. 5. Hogrefe, H.H., Griffth, J.P., Rossmann, M.G., Goldberg, E. Characterization of the antigenic sites on the refined 3-A resolution structure of mouse testicular lactate dehydrogenase C,. J . Biol. Chem. 262:13155-13162, 1987. 6. Grau, V.M., Trommer, W.E., Rossmann, M.G. Structure of the active ternary complex of pi heart lactate dehydrogenase with S-lac-NAD’ at 2.7 resolution. J . Mol. Biol. 151,289-307, 1981. 7. Piontek, K., Chakrabarthi, P., Schar, H.P., Rossmann, M.G., Zuber, H. Structure determination and refinement of Bacillus stearothermophilus lactate dehydrogenase. Proteins 734-92, 1990. 8. Hensel, R., Mayr, U., Yang, C.Y. The complete primary structure of the allosteric L-lactate dehydrogenase from Lactobacillus casei. Eur. J. Biochem. 134503-511, 1983. 9. Amelunxen, R.E., Murdock, A.L. Mechanisms of thermophily. CRC Crit. Rev. Microbiol. 6343-393, 1978. 10. Zuber, H. Structure and function of enzymes from thermophic microrganisms. In: “Strategies of Microbial Life in Extreme Environments,” Life Sciences Research Report 13, Dahlem Workshop Report, (Shilo, M., ed). Deerfield Beach, F1: Verlag Chemie International, 1979:393-415. 11. Schar, H.P., Zuber, H. Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria. I. isolation and characterization of lactate dehydroge-

1

(A’ldalton)

nases from thermophilic and mesophilic bacilli. Hoppe Seyler’s Z. Physiol. Chem. 360:795-807, 1979. 12. Zuber, H. Temperature adaptation of lactate dehydrogenase: Structural, functional and genetic aspects. Biophys. Chem. 29:171-179, 1988. 13. Clarke, A.R., Evington, J.R.N., Dunn, C.R., Atkinson, T., Holbrook, J.J. The molecular pathway by which fructose 1,6-bisphosphate induces the assembly of a bacterial lactate dehydrogenase. Biochim. Biophys. Acta 870:112-126, 1986. 14. Barstow, D.A., Clarke, A.R., Chia, W.N., Wigley, D., Sharman, A.F., Holbrook, J.J., Atkinson, T., Minton, N.P. Cloning, expression and complete nucleotide sequence of the Bacillus stearothermophilus L-lactate dehydrogenase gene. Gene 4647-55, 1986. 15. Zulli, F., Weber, H., Zuber, H. Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, VI. Nucleotide sequences of lactate dehydrogenase genes from the thermophilic bacteria Bacillus stearothermophilus, B. caldolyticus and B. caldotenax. Biol. Chem. 368:1167-1177, 1987. 16. Wirz, B., Suter, F., Zuber, H. Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria I11 The primary structure of thermophilic lactate dehydrogenase from Bacillus stearothermophilus. Hydroxylamine-, o-iodosobenzoic acid- and tryptic-fragments. The complete amino-acid sequence. Hoppe Seyler’s Z. Physiol. Chem. 364:893-909, 1983. 17. Clarke, A.R., Atkinson, T., Campbell, J.W., Holbrook, J.J. The assembly mechanism of the lactate dehydrogenase tetramer from Bacillus stearothermophilus; the equilibrium relationships between quaternary structure and the binding of fructose 1.6-bisphosphate,NADH and oxamate. Biochim. Biophys. Acta 829:387-396, 1985. 18. Clarke, A.R., Wigley, D.B., Chia, W.N., Barstow, D., Atkinson, T., Holbrook, J.J. Site-directed mutagenesis reveals role of mobile arginine residue in lactate dehydrogenase catalysis. Nature (London) 324599-702, 1986. 19. Clarke, A.R., Smith, C.J., Hart, K.W., Wilks, H.M., Chia, W.N., Lee, T.V., Birktoft, J.J., Banaszak, L.J., Barstow, D.A., Atkinson, T., Holbrook, J.J. Rational construction of a 2-hydroxyacid dehydrogenase with new substrate specificity. Biochem. Biophys. Res. Comm. 148:15-23, 1987. 20. Hart, K.W., Clarke, A.R., Wigley, D.B., Waldman, A.D.B., Chia, W.N., Barstow, D.A., Atkinson, T., Jones, J.B., Holbrook, J.J. A strong carboxylate-arginine interaction is important in substrate orientation and recognition in lactate dehydrogenase. Biochim. Biophys. Acta 914294-298. 1987. 21. Wilks, H.M., Hart, K.W., Feeney, R., Dunn, C.A., Muirhead, H., Chia, W.N., Barstow, D.A., Atkinson, T., Clarke, A.R., Holbrook, J.J. A specific, highly active malate dehydrogenase by redesign of a lactate dehydrogenase framework. Science 242:1541-1544, 1988. 22 Luyten, M.A., Bur, D., Wynn, H., Parris, W., Gold, M., Friesen, J.D., Jones, J.B. An evolution of the substrate specificity, and of its modification by site-directed mutagenesis, of the cloned L-lactate dehydrogenase from Bacillus stearothermophilus. J. Am. Chem. SOC.111:68006804, 1989. 23 Luyten, M.A., Gold, M., Friesen, J.D., Jones, J.B. On the effects of replacing the carboxylate-binding arginine-171 by hydrophobic tyrosine or tryptophan residues in the Llactate dehydrogenase from Bacillus stearothermophilus. Biochem. 2833605-6610, 1989.

CRYSTALLIZATION OF BSLDH MUTANTS 24. Bur, D., Clarke, T., Friesen, J.D., Gold, M., Hart, K.W., Holbrook, J.J., Jones, J.B., Luyten, M.A., Wilks, H.M. On the effects of specificity of Thr246-Gly mutation in L-lactate dehvdropenase of Bacillus stearothermoohilus. Biochem. B;oph$. Res. Comm. 16159-63, 1989.’ 25 Wilks, H.M., Halsall, D.J., Atkinson, T., Chia, W.N., Clarke, A.R., Holbrook, J.J. Design for a broad substrate specificity keto acid dehydrogenase. Biochem. 29: 8587-8591, 1990. 26. Matthews, B.W. Solvent content ofprotein crystals. J . Mol. Biol. 33:491-497, 1968. 27. Sakabe, N. A focusing weissenberg camera with multi-

161

layer line screens for macromolecular crystallography. J . Appl. Cryst. 16542-547, 1983. 28. Miyahara, J., Takahashi, K., Amemiya, Y., Kamiya, N., Satow, Y. A new type of X-ray area detector utilizing laser stimulated luminescence. Nucl. Instrum. Methods A246: 572-578, 1986. 29. Higashi, T. The processing of diffraction data taken on a screenless weissenberg camera for macromolecular crystallography. J. Appl. Cryst. 22:9-18, 1989. 30. Rossmann, M.G. ed. The molecular replacement method. A collection of papers on the use of non-crystallographic symmetry. New York Gordon & Breach, 1972.