.J. MoE. Biol. (1985) 184. 311-318 Low Resolution Crystal Structure of Muconolactone Isomerase A Decamer with a 5-fold Symmetry Axis Bradley A. Katz?, David Ollis and Harold W. Wyckoff Department of Molecular Biophysics and Biochemistry, Yale University, Xeu! Haven, CT 06511, I’.A.d (Received 10 December 1984, and in revised form 5 A’arch, 1985) Muconolactone isomerase from Pseudomonas putida crystallizes from sodium sulfate solution in space group P2, (a = 65.84 A, b = 105.70 A, c = 77.20 A, /? = 90.5”) with ten 11.OOO M, subunits per asymmetric unit. The 7 A resolution crystal structure was solved by single isomorphous replacement followed by IO-fold symmetry averaging. The decameric enzyme has an uncommon non-crystallographic &fold symmetry axis and a large cavit,y in itIs (aenter. 1. Introduction As part of a study of structural relationships among the bacterial enzymes involved in the /I- ketoadipate pathway (Yeh et al., 1980; Yeh & Ornston, 1980; Ornston & Yeh, 1979), we have begun a program to determine crystallographically the three-dimensional structures of the key enzymes involved. Among the many reactions in this catabolic pathway, which mediates dissimilation of aromatic growth substances to tricarboxylic acid intermediates, is a lactonization reaction carried out by cis,ris-muconate-lactonizing enzyme (EC 5.5.1.1). followed by an intralactonic rearrange- ment of a double bond catalyzed by muconolactone isomerase (EC 5.3.3.4). As a basis for investigations attempting to probe the genetic evolution of the /?- ketoadipate pathway by comparison of its enzyme structures, and also for mechanistic studies of the diverse biochemical reactions exemplified by its many enzymes, X-ray diffraction studies of two consecutive enzymes in the pathway, cis,cis- muconate-lactonizing enzyme (Goldman et al.. 1985) and muconolactone isomerase, have been undertaken. We describe here the 7 A resolution crystal structure of muconolactone isomerase (Pseudomonas putida. strain PRS2000), an oligomer showing unusual molecular symmetry. Muconolactone isomerase catalyzes the con- t Author to whom all correspondence should be addressed. Present address: Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080. 1T.S.A. - version of muconolactone to fi-ketoadipate enol lactone. It consists of ten 11,000 IW, subunits, each composed of 97 amino acid residues (Meagher & Ornston. 1973; Yeh & Ornston, 1980). Procedures for purification and preparation of microcrystalline muconolactone isomerase from distilled wa,ter or from ammonium sulfate solution have been described (Ornston, 1966). The oligomer molecular weight’ inferred from sedimentation equilibrium ultracentrifugation (111,000) or from sodium dodecyl sulfate/polyacrylamide gel electrophoresis followmg dimethyl suberimidate cross-linking (124,000), suggested either a dreamer or a dodecamer in solution (Parke. 1979). 2. Experimental Procedures and Results Crystallization by vapor diffusion at 4°C from 80,b saturated sodium sulfate solution buffered with MESS (pH 6.0) affords large monoclinic rods (space group P2,; a = 65.84 A, b = 105.70 *&, c = 77.20 A, /I = 90*5”)$. which diffract t,o beyond 2.8 a IAbbreviations used: MES: N-morpholinoethane sulfonate; pCMBS: p-chloromercuribenzene sulfonate. $ We have encountered :! closely similar monoclinic space groups with all lattice parameters identical except for /I. which can assume a value of either 90.5” or of H9.5”, for crystals grown under these conditions. The polymorphism was inadvertently discovered by the observation that data sets collected on different crystals (all indexed with /I = 90.5”) could be merged successfully only if some of the datasets were re-indexed by reversing t,he handedness of the unit cells of the corresponding crvstals.