Acc. Chem. Res. 2001, 34, 145-157 similar three-dimensional architectures. In each protein, The Crotonase Superfamily: a common structural strategy is employed to lower the Divergently Related Enzymes free energies of chemically similar intermediates. Catalysis of the divergent chemistries is accomplished by both That Catalyze Different retaining those functional groups that catalyze the com- mon partial reaction and incorporating new groups that Reactions Involving Acyl direct the intermediate to new products. Indeed, as a Coenzyme A Thioesters specific example, the enolase superfamily has served as a paradigm for the study of catalytically diverse superfami- HAZEL M. HOLDEN,*,³ lies.3 The active sites of proteins in the enolase superfamily MATTHEW M. BENNING,³ are located at the interfaces between two structural TOOMAS HALLER,² AND JOHN A. GERLT*,² motifs: the catalytic groups are positioned in conserved Departments of Biochemistry, University of Illinois, regions at the ends of the â-strands forming (R/â) 8-barrels, Urbana, Illinois 61801, and University of Wisconsin, while the specificity determinants are found in flexible Madison, Wisconsin 53706 loops in the capping domains formed by the N- and Received August 9, 2000 C-terminal portions of the polypeptide chains. While the members of the enolase superfamily share similar three- ABSTRACT dimensional architectures, they catalyze different overall Synergistic investigations of the reactions catalyzed by several reactions that share a common partial reaction: abstrac- members of an enzyme superfamily provide a more complete tion of an R-proton from a carboxylate anion substrate understanding of the relationships between structure and function than is possible from focused studies of a single enzyme alone. to form an enolic intermediate that is stabilized by The crotonase (or enoyl-CoA hydratase) superfamily is such an interactions with an essential divalent metal ion. By virtue example whereby members catalyze a wide range of metabolic of this stabilization, the intermediates are kinetically reactions but share a common structural solution to a mechanistic competent. Depending upon the overall reaction, how- problem. Some enzymes in the superfamily have been shown to display dehalogenase, hydratase, and isomerase activities. Others ever, the enolic intermediates partition to products via 1,1- have been implicated in carbon-carbon bond formation and proton transfer (racemization or epimerization) or â-e- cleavage as well as the hydrolysis of thioesters. While seemingly limination (dehydration or deamination). unrelated mechanistically, the common theme in this superfamily is the need to stabilize an enolate anion intermediate derived from The crotonase superfamily described here displays an acyl-CoA substrate. This apparently is accomplished by two mechanistic diversity at least equal to, or perhaps even structurally conserved peptidic NH groups that provide hydrogen greater than, that of the enolase superfamily. Indeed, the bonds to the carbonyl moieties of the acyl-CoA substrates and form documented reactions catalyzed by members of this an ªoxyanion holeº. superfamily include dehalogenation, hydration/dehydra- tion, decarboxylation, formation/cleavage of carbon- carbon bonds, and hydrolysis of thioesters. Additionally, Introduction members of the crotonase superfamily are involved in a The study of enzyme superfamilies whose members wide range of metabolic pathways. The first structure to catalyze different overall reactions allows a more complete be solved for a member of this superfamily was 4-chlo- understanding of the relationships between structure and robenzoyl-CoA dehalogenase from Pseudomonas sp. strain function than is possible from studies of individual CBS-3.4 This investigation was followed by the elegant proteins.1,2 Members of such mechanistically diverse structural analyses of the rat enoyl-CoA hydratase (com- superfamilies are thought to be related by divergent monly referred to as crotonase, hence the name of the evolution from a common progenitor, therefore displaying superfamily)5,6 and the rat ∆3,5,∆2,4-dienoyl-CoA isomerase.7 low levels of sequence identity/similarity but adopting Very recently the molecular motif of an additional member of the crotonase superfamily has been determined, namely methylmalonyl-CoA decarboxylase from Escherichia coli.8 Hazel M. Holden received an A.B. degree in chemistry at Duke University (1977) and a Ph.D. in biochemistry at Washington University in St. Louis (1982). She is This X-ray analysis of methylmalonyl-CoA decarboxylase currently a Professor in the Biochemistry Department at the University of was especially enlightening in that several of the unex- Wisconsin, Madison. pected changes in three-dimensional structure could not Matthew M. Benning received a B.A. degree in chemistry and physics at have been predicted on the basis of amino acid sequence Augustana College (1983) and a Ph.D. in chemistry at Northern Illinois University alignments alone. (1988). He is currently an Associate Scientist at the University of Wisconsin, Madison. Here we describe the overall three-dimensional struc- tures and active site geometries of 4-chlorobenzoyl-CoA Toomas Haller received a B.S. degree in biochemistry at the University of Tartu 3,5 2,4 (Estonia) in 1996. He is currently a graduate student in the Department of dehalogenase, crotonase, ∆ ,∆ -dienoyl-CoA isomerase, Biochemistry at the University of Illinois. * Address correspondence to these authors. H.M.H.: E-mail John A. Gerlt received a B.S. degree in biochemistry at Michigan State University [email protected], fax 608-262-1319, phone 608-262-4988. (1969) and A.M. (1970) and Ph.D. (1974) degrees in biochemistry and molecular J.A.G.: E-mail [email protected], fax 217-265-0385, phone 217-244-7414. biology at Harvard University. He is a Professor and the Head in the Department ² University of Illinois. of Biochemistry at the University of Illinois, Urbana-Champaign. ³ University of Wisconsin. 10.1021/ar000053l CCC: $20.00 2001 American Chemical Society VOL. 34, NO. 2, 2001 / ACCOUNTS OF CHEMICAL RESEARCH 145 Published on Web 12/01/2000 The Crotonase Superfamily Holden et al. FIGURE 1. Enolate anion of a CoA thioester. FIGURE 2. 4-Chlorobenzoic acid degrading pathway in Pseudomonas sp. strain CBS-3. FIGURE 3. Catalytic mechanism for 4-chlorobenzoyl-CoA dehalogenase. and methylmalonyl-CoA decarboxylase in the context of referred to as PCBs.9 Some bacteria, such as Pseudomonas their known biochemical properties. The common theme sp. strain CBS-3,10 are able to utilize these various chlo- behind all of the reactions catalyzed within this super- rinated compounds as their sole source of carbon via the family is the stabilization of an enolate anion intermediate 4-chlorobenzoate degrading pathway illustrated in Figure of acyl-CoA substrates (Figure 1) by two structurally 2.11,12 Of particular interest is the second enzyme in the conserved peptidic NH groups that form an ªoxyanion pathway, namely 4-chlorobenzoyl-CoA dehalogenase, here- holeº. The goal of this Account is to emphasize our current after referred to simply as dehalogenase. This enzyme has state of knowledge regarding both the similarities and the attracted significant research attention due to its quite differences exhibited by members of the crotonase su- specialized mode of catalysis, as indicated in Figure 3.13-20 perfamily. According to all presently available biochemical data, the reaction mechanism of the dehalogenase proceeds via 4-Chlorobenzoyl-CoA Dehalogenase from attack of the side-chain carboxylate group of Asp 145 on Pseudomonas sp. Strain CBS-3 the benzoyl ring of the substrate at position C(4), leading During the latter part of the twentieth century, synthetic to the formation of a Meisenheimer complex. Displace- chlorinated organic compounds accumulated in the en- ment of the halide results in an arylated enzyme inter- vironment as a result of both commercial production and mediate, which subsequently is hydrolyzed by an activated careless waste disposal. The chlorobenzoic acids, for water molecule. example, now found in the environment often result from An X-ray crystallographic analysis of the dehalogenase the microbial degradation of polychlorinated biphenyls, from Pseudomonas sp. strain CBS-3 was initiated in an 146 ACCOUNTS OF CHEMICAL RESEARCH / VOL. 34, NO. 2, 2001 The Crotonase Superfamily Holden et al. FIGURE 4. Ribbon representation of 4-chlorobenzoyl-CoA dehalogenase. The quaternary structure of the enzyme is trimeric as indicated in (a) with the three subunits color-coded in red, green, and blue. The positions of the active sites in each subunit are indicated by the 4-hydroxybenzoyl-CoA ligands drawn in ball-and-stick representations. Pairs of active sites are separated by approximately 42 Å. The crown of three tryptophan residues that play a role in maintaining the trimeric interface of the dehalogenase is displayed in a ball-and-stick representation. An individual subunit is shown in stereo in (b) with the R-helices and â-strands displayed in blue and red, respectively. As indicated by the orange sphere, a cation (most likely a calcium ion) is positioned between the two R-helices connecting the N-terminal and trimerization domains. X-ray coordinates for 4-chlorobenzoyl-CoA dehalogenase were determined in the Holden laboratory and can be obtained from the Protein Data Bank (1NZY). effort to address several key structural features concerning susceptible to nucleophilic attack