Crystallization and Preliminary Crystallographic Analysis of Aspartate-p-semialdehyde Dehydrogenase from Escherichia coli Gitay Kryger, Gregory A. Petsko Departments of Chemistry and Biochemistry, Rosen&e1 (Jenter Brandeis University, Waltham, MA 022.54-9110, U.S.A.

Jun Ouyang and Ronald E. Viola? Department

of Chemistry,

University

(Received 12 May

of Akron,

Akron,

1992; accepted 6 July

OH 44325-3601,

U.8.A.

1992)

Aspartate-P-semialdehyde dehydrogenase catalyzes the NADPH-mediated reductive dephosphorylation of B-aspartylphosphate at a branch point in the biosynthesis of several amino acids. The enzyme from Escherichia coli has been crystallized by the vapor diffusion method from Tris buffer (pH W5) using polyethylene glycol 4000 as a precipitant. The crystals are orthorhombi: and have0 the symmetry of space group P222,, with unit cell dimensions of a = 177.8 A, b = 599 A, c = 118.65 A, and u = p = y = 90”. The dimensions and space group are indicative of two enzyme dimers (40 kDa per subunit) in the asymmetric unit.0 The crystals show strong diffraction, and a native data set has been collected to 2.5 A resolution. Keywords: protein crystallization;

enzyme; aspartate-/?-semialdehyde Escherichia coli

The enzyme aspartate-fi-semialdehyde dehydrogenase (ASA DHS, E.C.1.2.1.11) is a pyridine nucleotide (NADP)-linked dehydrogenase that is encoded by the asd gene in Escherichia coli. The reaction catalyzed by this enzyme is located at a key branch point (Fig. 1) in the aspartate pathway of amino acid biosynthesis (Cohen, 1983). The primary structure of the enzyme has been deducted from the nucleotide sequence of the gene (Haziza et al., 1982). The enzyme exists as a homodimer, with each subunit containing 367 amino acids, and a molecular weight, calculated from the amino acid sequence, of 39,950. The asd gene has been cloned and incorporated into an expression system (Preiss et aE., 1982). The mechanism of ASA DH has been examined by kinetic and chemical modification studies (Karsten & Viola, 1991), and by sitedirected mutagenesis (Karsten & Viola, 1992). A preliminary crystallization of E. co&i ASA DHhas previously been reported (Thierry et al., 1980);

dehydrogenase;

however, this work did not progress to a high resolution structure of the enzyme due to the unreproducibility of the crystallization. The asd gene was incorporated into a multicopy plasmid (pGEM) which was transformed into JM109 cells to provide the source for ASA DH. The enzyme was purified using a previously published purification scheme (Karsten & Viola, 1991) to yield 20 mg of purified enzyme from the cells grown in one litre of minimal media. The enzyme was determined to be >95% pure as judged by densitometer scans of SDS/polyacrylamide gel electrophoresis gels. The specific activity of 100 units/mg protein at pH 8.6 is comparable to the highest values that have been reported. The enzyme stock solutions for the crystallization trials contained approximately 10 mg protein/ml in 10 mM-Hepes buffer (pH 8.0) with 1 mM each of EDTA, dithiothreitol, and sodium azide. Crystals of ASA DH were grown by using both the hanging-drop and sitting-drop techniques. The protein stock solution was added to the crystallization medium, which contained 19 to 20% polyethylene glycol 4000, 625 to 0.30 M-MgCl, and 61 M-Tris buffer (pH &5) to give a final protein

t Author to whom all correspondence should be addressed. $ Abbreviations used: ASA DH, aspartateP-semialdehyde dehydrogenase. 300 0022-2836/92/2103OC42

$08.00/O

(~3 1992 Academic

Press Limitetl

Crystallization

NH’

0

AK b

- OOC-CH,-b&00-

NH;

- O,P-0-&CH,-&H-COO-

I OH

NH’

H,&CH,-kHfCO0

HSD .

ASA-DH

0 NH; II HC-CH$H-COO.

c Methionine Threonine lsoleucine

Lysine

Figure 1. Asp&ate pathway of amino acid biosyntheis in E. coli. AK, aspartokinases; ASA DH. aspartatedehydrogenase; HSD. homoserine j-semialdehyde dehydrogenases. concentration of 4.6 mg/ml. Crystals were obtained at room temperature in one to two days; however, these crystals are somewhat disordered. Successive seedings at room temperature did lead to small, but highly ordered crystals. Crystallization trials at 4°C gave larger, highly ordered crystals of at least @8 mm in each dimension in about one week. These crystals are quite fragile under mild physical stress, but are stable in the X-ray beam for more than three days. The crystals diffract quite strongly. A native data set has been collected at room temperature to 2.5 A resolution (1 A = 0.1 nm), with better than 75% completeness in the outermost. shells and reasonable internal statistics, by using a double Xuong multiwire area detector system on a Rigaku RU-200 rotating anode X-ray source. The space group was determined from diffractometer measurements to be P222,, with systematic absences observed on only one axis. The unit cell perameters wer? determine,d to be: a = 177.8(l) A, b = 599(2) A, and c = 11&65(2) A. Using the calculated subunit molecular weight of 39,950. the unit cell dimensions are consistent with two enzyme dimers in the asymmetric unit. Although the space group is the same as that reported in the earlier crystallographic investigation of this enzyme (Thierry et aE., 1980), t)he unit cell

301

Xotes

dimensions are different and the content of the asymmetric unit is doubled. These crystals can be grown reproducibly and appear suitable for the determination of the structure of aspartate-psemialdehyde dehydrogenase by X-ra,y diffraction.

This work was supported by research grants. from the Xational Institutes of Health (GM26788) to G.A.P., and the Charles Culpeper Foundation to R.E.V. The authors thank Dr Jack Preiss for supPlying the as&containing plasmid. Acknowledgement, is given to the following members of the Struct’ural Biology Department’ at the Weizmann 1nst)itute in Rehovot. Israel. where these crystallization t)rials were conducted: to Professor A. Yonath who made this work possible by providing all attainable material and spiritual support, to Dr F. Frolow for his guidance and practical help in each step, to Y. Halfon for his highly skilled technical help, and to Dr S. Weinstein, M. Laschever and G. Tdan for technical assistance.

References Cohen G. N. (1983). The common pathway to lysine, methionine, and threonine. In Amino Acids: Biosynthesis

and

Genetic

Regulation

(Herrmann,

K. M. & Somerville, R. L., eds), pp. 147-171, Addison-Wesley, Reading, MA. Haziza, C., Stragier, P. & Patte, J. C. (1982). Xucleotide sequence of the asd gene of Eseherichia coli: absence of a typical attenuation signal. EMBO J. 1, 379-384. Karsten, W. E. & Viola, R. E. (1991). Chemical and kinetic mechanism of aspartatt-p-semialdehyde dehydrogenase from Escherichia coli. Biochim. Biophys. Acta, 1077, 209-219. Karsten, W. E. & Viola, R. E. (1992). Identification of an essential cysteine in the reaction catalyzed by aspartate-b-semialdehyde dehydrogenase from Escherichia, coli. Biochim. Biophys. Acta. 1121, 234-238. Preiss. J., Mazelis. M. & Greenberg, E. (1982). Cloning of the aspartatt-P-semialdehyde dehydrogenase structural gene from E. coli K12. Cflrr. Microbial 7, 263-268. Thierry, J. C., Moras, D., Eid, P. t Hirt’h. C. (1980). Crystallization of E. coli aspartate-b-semialdehyde dehydrogenase. Biochimie, 62, 739-740.

Edited by B. W. klatthews