Prorein Science zyxwvutsrqponm (1997), 6:1774-1776. Cambridge University Press. Printed in the USA. Copyright zyxwvutsrqp 0 1997 The Protein Society zyxwvutsrqp FOR zyxwvutsr THE RECORD Crystallization of 5-aminolaevulinic acid dehydratase from Escherichia coli and saccharomyces cerevisiae and preliminary X-ray characterization of the crystals P.T. ERSKINE,' N. SENIOR,* S. MAIGNAN,'.' J. COOPER,' R. LAMBERT,' G. LEWIS,' P. SPENCER,'.' S. AWAN,* M. WARREN,2 I.J. TICKLE,3 P. THOMAS," S.P. WOOD,' AND P.M. SHOOLINGIN-JORDAN' zyxwvutsr ' Department of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, 2Laboratory of Molecular Genetics, Institute of Ophthalmology, University College London, Bath Street, London, 'Department of Crystallography, Birkbeck College, University of London, Malet Street, London, WClE 7HX, United Kingdom 4Zeneca Agrochemicals, Jealotts Hill Research Station, Bracknell, Berkshire, RG12 6EY. United Kingdom (RECEIVED February 14, 1997; ACCEPTED April 18, 1997) Bassett Crescent East, Southampton, SO16 7PX, United Kingdom EClV 9EL, United Kingdom Abstract: 5-Aminolaevulinic acid dehydratase (ALAD) catalyzes the formation of porphobilinogen from two molecules of 5-amino- laevulinic acid. Both Escherichia coli and Saccharomyces cere- visiae ALADs are homo-octameric enzymes which depend on Zn2+ for catalytic activity and are potently inhibited by lead ions. The E. coli enzyme crystallized in space group 1422 (unit cell dimen- sions a = b= 130.7 A, c = 142.4 A). The best crystals were obtained in the presence of the covalently bound inhibitor laevu- linic acid. The yeast enzyme (expressed in E. coli) crystallized in the same space group (1422) but with a smaller unit cell volume (a = b = 103.7 A, c = 167.7 A). High resolution synchrotron data sets were obtained from both E. coli and yeast ALAD crystals by cryocooling to 100 K. Keywords: cryocooling; crystallization; 5-aminolaevulinic acid de- hydratase, porphobilinogen synthase The enzyme 5-aminolaevulinate dehydratase (ALAD, E.C.4.2.1.24), also referred to as porphobilinogen synthase, catalyzes an early step in the biosynthesis of tetrapyrroles involving the condensation of two molecules of 5-aminolaevulinic acid (ALA) to form the pyrrole porphobilinogen (see Fig. 1). Four molecules of porpho- bilinogen are condensed in a reaction catalyzed by porphobilinogen Reprint requests to J. Cooper, Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Cres- cent East, Southampton, SO16 7PX, United Kingdom; e-mail: jbc2@soton. ac.uk. 5Present address: Laboratoire de Biologie Structurale, Universite de Paris- Sud, UPR 9063 CNRS. 91 198 Gif Sur Yvette Cedex, France. 'Present address: Center for Biological NMR, Department of Chemistry, Texas A & M University, College Station, Texas 77843. deaminase (Louie et al., 1992, 1996) to form a linear tetrapyrrole, which is subsequently cyclized and rearranged to give the cyclic tetrapyrrole uroporphyrinogen zyxw 111. As precursors for haem, chlo- rophyll, and vitamin BIZr which play critical roles in respiration, photosynthesis, and metabolism, tetrapyrroles and their derivatives are essential biological molecules (for reviews, see Warren & Scott, 1990; Jordan, 1991, 1994; Jaffe, 1995). The hereditary deficiency of functional dehydratase in humans is associated with the reces- sive genetic disease Doss or ALA dehydratase porphyria. The se- vere neurological symptoms of this disease may be due to the accumulation of 5-aminolaevulinic acid, which mimics the inhib- itory neurotransmitter y-aminobutyric acid (GABA). Inhibition of ALAD by lead ions is one of the major manifestations of acute lead poisoning, which leads to anemia and often psychosis. An- other intriguing function of ALAD is that it appears to be identical to the CF-2 inhibitory component of the proteasome complex (Guo et al., 1994), which may represent an interesting example of gene sharing. 5-ALA dehydratases largely occur as octameric enzymes con- sisting of identical subunits, each with a molecular mass of COOH A-slde P-s~de SOOH Fig. 1. The condensation reaction catalyzed by 5-aminolaevulinic acid de- hydratase (ALAD). 1774