Journal of Molecular Biology
Regular articleCrystallographic analysis of the reaction pathway of Zoogloea ramigera biosynthetic thiolase1
Introduction
Thiolases are ubiquitous enzymes which play key roles in many vital biochemical pathways Gehring and Lynen 1972, Thompson et al 1989, including the β-oxidation pathway of fatty acid degradation and various biosynthetic pathways. Members of the thiolase family can be divided into two categories: degradative thiolases (E.C. 2.3.1.16) and biosynthetic thiolases (E.C. 2.3.1.9). Degradative thiolases, also known as 3-ketoacyl-CoA thiolases, catalyze the thiolytic cleavage of 3-ketoacyl-CoA molecules to yield acetyl-CoA and a shortened acyl-CoA species (Figure 1). This is the fourth and final step in the fatty acid β-oxidation pathway, which is responsible for the extraction of the metabolic energy stored in fats and fatty acids (Kunau, et al., 1995). Biosynthetic thiolases, also known as acetoacetyl-CoA thiolases, catalyze the biological Claisen condensation of two acetyl-CoA molecules to form acetoacetyl-CoA (Walsh, 1979). This is one of the fundamental categories of carbon skeletal assembly patterns in biological systems and is the first step in many biosynthetic pathways. In higher eukaryotes, these include the pathways leading to the synthesis of ketone bodies, cholesterol and steroid hormones. Certain prokaryotes, such as Zoogloea ramigera, rely on biosynthetic thiolases for the synthesis of poly-3-d-hydroxybutyrate as their principal energy storage molecule (Masamune et al., 1989a). The physiological importance of thiolases is illustrated by the severe and usually lethal phenotypes of patients with deficient degradative Tyni et al 1997, Kamijo et al 1997 or biosynthetic Hartlage et al 1986, Fukao et al 1998 thiolases.
All thiolases are sequence related and are thought to employ the same reaction mechanism, in which a covalent intermediate is formed between the reactive cysteine residue of the enzyme and an acyl group Gehring et al 1968, Gehring and Lynen 1972, Gilbert et al 1981. The biosynthetic thiolase reaction is thermodynamically very unfavorable (Gilbert et al., 1981), but all thiolases catalyze the reaction in both directions. The degradative thiolases have a broad substrate specificity, catalyzing the reaction of fatty acid molecules of various lengths. In contrast, biosynthetic thiolases only catalyze the formation of acetoacetyl-CoA from two molecules of acetyl-CoA or the reverse reaction.
With respect to kinetic properties, the best characterized degradative thiolase is mitochondrial pig heart tetrameric thiolase, classically referred to as thiolase I (Gilbert et al., 1981). Thiolase I activity has a broad pH optimum with optimal activity between pH 6 and pH 9. The best characterized biosynthetic thiolase is the bacterial tetrameric thiolase from Zoogloea ramigera, referred to here as biosynthetic thiolase. This thiolase was first purified by Tomita et al. (1981). In this first characterization it was established that the pH optimum for the thiolytic and condensation reactions are respectively 8.5 and 7.5. The kinetic data for these two thiolases are rather different, as can be seen in Table 1. The kinetic differences are of two kinds: (i) large differences in kcat; and (ii) differences with respect to the rate-limiting step. Table 1 shows that biosynthetic thiolase is much more efficient than thiolase I. Also, the rate of hydrolysis of the acetylated intermediate of biosynthetic thiolase is much greater than that of thiolase I (Table 1). The differences in rate-limiting steps are visualized in Figure 1. For thiolase I, the rate-limiting steps are the steps by which the covalent intermediate is formed, whereas for the biosynthetic thiolase, the steps by which the covalent intermediate is degraded are the rate-limiting steps Thompson et al 1989, Gilbert et al 1981, Masamune et al 1989a.
Enzyme kinetics, active site labeling and site-directed mutagenesis experiments with the biosynthetic thiolase of Z. ramigera allowed two conserved cysteine residues to be identified as being important for catalysis Thompson et al 1989, Palmer et al 1991. On the basis of these studies, a two step ping-pong reaction mechanism was proposed (Masamune et al., 1989b). In the first step of the reaction Cys89 (Thompson et al., 1989) nucleophilically attacks the acyl-CoA substrate, leading to the formation of the covalent acyl-CoA intermediate. In the second step, the addition of acetyl-CoA (in the biosynthetic reaction) or CoA (in the degradative reaction) to the acyl-enzyme intermediate triggers the release of product from the enzyme. This step is facilitated by a second cysteine residue, Cys378 in biosynthetic thiolase, which is believed to be involved in proton exchange Palmer et al 1991, Williams et al 1992, Modis and Wierenga 1999.
Most thiolases are tetrameric, but peroxisomal degradative thiolases are dimeric (Kunau et al., 1995). The crystal structures of two thiolases have been reported previously, the first was the dimeric peroxisomal yeast thiolase (involved in the fatty acid β-oxidation pathway) and the second a bacterial tetrameric biosynthetic thiolase (involved in the synthesis of poly-3-d-hydroxybutyrate). The crystal structure of the yeast thiolase, referred to henceforth as degradative thiolase, was refined at 1.9 Å resolution (Mathieu et al., 1997). The enzyme is unliganded in this structure. The kinetic properties of yeast degradative thiolase are similar to other degradative thiolases (Miyazawa et al., 1981). The structure of biosynthetic thiolase, also refined at 1.9 Å resolution, reveals an acyl-enzyme intermediate in complex with CoA (state II of Figure 1). The sequence identity between the yeast degradative and bacterial biosynthetic thiolases is about 38 %. The two enzymes share the same fold. Each subunit consists of two core domains plus a large loop domain, (residues 119-249 in biosynthetic thiolase) inserted in the N-terminal core domain. Most of the residues interacting with CoA are in the loop domain (Modis & Wierenga, 1999). The two core domains form a five-layered αβαβα structure; the central α-layer is made of two buried α-helices which follow immediately after the catalytic Cys89 and His348, respectively. Thiolase subunits tightly associate to form dimers (Mathieu et al., 1997); the tetrameric thiolase is built up of two dimers which are flexibly associated via four interacting loops (residues 123-141), one from each subunit (Figure 2; Modis & Wierenga, 1999). These loops protrude out of the bulk of the two dimers, in the center of the tetramer. The four active sites are near these loops, facing the inter-dimer space (Figure 2). In degradative thiolase the active site pocket is large enough to accommodate long-chain fatty acids, but in biosynthetic thiolases the binding pocket is much smaller, due to the conformation of residues 142-148 of the loop domain (Modis & Wierenga, 1999).
Here, we report the structures of three more complexes of the biosynthetic thiolase. A comparison of these structures with the two other known thiolase structures (Table 2) yields important new insights into the thiolase reaction mechanism.
Section snippets
Results and discussion
Three new crystal structures were obtained of biosynthetic thiolase from Z. ramigera (Table 2): unliganded, in complex with CoA, and in complex with the second reaction intermediate in the biosynthetic reaction (state III in Figure 1). The crystals were obtained by co-crystallization and soaking experiments (Table 3). The initial models of these structures were derived from the previously published structure of the Z. ramigera thiolase (Modis & Wierenga, 1999). The models for the three
Concluding remarks
The new structural information is in good agreement with the sequence of steps outlined in Figure 1. In the first step, Cys89 is acetylated by acetyl-CoA. In the proposed mechanism His348 accepts a proton from the sulfur atom of Cys89 in the acetylation step, and the sulfur atom of Cys378 donates its proton to the sulfur atom of the leaving group, namely CoA. The deprotonated side-chain of Cys378 is probably stabilized by the extensive hydrogen bonding network, outlined in Figure 7; it is
Crystallization, data collection and refinement
Biosynthetic thiolase from Z. ramigera was expressed and purified from Escherichia coli as described by Thompson et al. (1989). Crystals of biosynthetic thiolase were grown using the hanging drop vapor diffusion method, as described by Modis & Wierenga (1999), in a mother liquor containing 0.9 M ammonium sulfate, 1 M lithium sulfate, 0.1 M sodium acetate (pH 5.0), 1 mM EDTA, 1 mM sodium azide and 1 mM reduced DTT. Crystals obtained under these conditions contained unliganded thiolase and
Acknowledgements
We thank Matti Saraste and Andreas Hönger for their support at EMBL-Heidelberg. We gratefully acknowledge the availability of beam time at DESY, Hamburg (beamline X31). We thank JoAnne Stubbe and Ute Müh for providing the expression construct of the biosynthetic thiolase. We are grateful to Daan van Aalten for his assistance during synchrotron data collection and to Drs Schmitz and Hiltunen for fruitful discussions.
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Present address: Y. Modis, Department of Biological Chemistry and Molecular Pharmacology and Howard Hughes Medical Institute, Harvard Medical School, Enders Research Building, 320 Longwood Avenue, Boston, MA 02115, USA