.J, Mol. Rio/. (19X2) 162, 419-444 Structure of 2-Keto-3-deoxy-6-phosphogluconate Aldolase at 24 A Resolution TIWSE: M. Ma\wmt. MAWOS H. H.u.wA$, A. Tr.~,r~uslir- ASI) LIXASZ I~~HIOI~A~ Department of Chemistry. Michigan State Imiver.city East Lansing. Mich. 48824, V.S.A (Received 19 April 1982, and in revised form 26 July 1982) The structure of d-keto-3-deoxy-6-phosphogluconate aldolase has been extended to 2.8 B resolution from 3.5 A resolution by multiple isomorphous replacement methods using three heavy-atom derivatives and anomalous Hijvoet differences t,o 6 A resolution ((m) = @72). Th e replacement phases were improved and refined by electron density modification proredures coupled wit)h inverse t’ransform phase angle calculations. A Kendrew model of the molecule was built. which contained all 225 residues of a recently determined amino acid sequence. whereas only 173 were accounted for at 3.5 i%resolution. The missing residues were found to be part of the interior of the molecule and not simply an appendage. The molecule folds to form an eight-strand +barrel strurtunl strikingly similar to triosephosphate isomerase. thp A-domain of ppruvate kinase and Taka amglase. M’ith a knowledge of the sequence. the nature of the interfaces of the two kinds of crystallographic trimers have been examined. from which it was concluded that the choice of trimers selected in the 3.5 A resolution work was probably correct for trimers in solution. The active site region has been established from the position of the Schiff base forming Lys144 but it has not been possible to confirm it conclusively in independent derivative experiments. An apparent, anotnaly exists in the location of Clu56 (about 25 A from Lys144). The latt,er has been reported to assist in catalysis. 1. Introduction The a-keto-/3-deoxycarboxylic acid aldolases (Wood, 1972), also called pyruvate- specific lyases (Meloche, 1981), emerged when a new hexose monophosphate pathway from glucose to pyruvate was discovered in Pseudomonas saccharophila (Entner & Doudoroff, 1952; MacGee & Doudoroff, 1954). The aldolase of this study, KDPGjI aldolase, is specific for 2-keto-3-deoxy-6-phosphogluconate and is utilized in other enzymic forms to produce a variety of acceptor aldehydes and pyruvate. The KDPG aldolase employed for the crystallographic work was isolated from Pseudomonas putida and crystallized with ammonium sulfate (Vandlen et al.. 1973 ; t Present address: Chemistry Division. Nuclear Research Center. l)rmocritos, Athens, Greece. 1 Present address: Department of Chemistr?, 1Tniversity of California, Berkeley. CA 94720, U.S.A. $ Permanent address: Department of Chemistry. Jagielionian I’niversity. Krakow. Poland. 1) Abbreviations used: KDPG. 2.k~to-3.droxv-B-phosphoglu~~nate: m.i.r.. multiple isomorphous replacement: r.m.s.. root-mean-square; TIMase. triosephosphate isomcrasr: I’Kase, pyruvate kinas?. 419 W2-2836/82/340419-26 $03.00/O CP 1982 Academic Press Inc. (London) Ltd.