Journal of Molecular Biology
Crystal Structure of Yeast Peroxisomal Multifunctional Enzyme: Structural Basis for Substrate Specificity of (3R)-hydroxyacyl-CoA Dehydrogenase Units
Introduction
In living organisms, quantitatively the major pathway for degrading fatty acids is β-oxidation, which consists of a set of four reactions operating at carbon 2 and 3 of acyl-CoA esters. To allow the β-oxidation of acyl groups with different chain lengths to proceed, many enzymes of β-oxidation have evolved as paralogues, showing specificity with respect to variants of acyl groups.1, 2
Yeasts and mammals possess a profound difference in completing the utilization of fatty acids. Mammals have two separate systems: peroxisomal and mitochondrial β-oxidation pathways, which use the same chemistry for sequential cleavage of the fatty acyl chains, but utilize different gene products for the purpose. Mammalian peroxisomal β-oxidation carries out partial chain-shortening of a wide variety of substrates, ranging from very long and long to medium straight-chain saturated and unsaturated fatty acids, as well as α-methyl-branched chain fatty acids and bile acid intermediates.3, 4 In mitochondria, long-chain fatty acids are β-oxidized by membrane-associated enzymes, whereas the short-chain fatty acids are oxidized by a set of soluble monofunctional enzymes, allowing the degradation of fatty acids to go all the way to acetyl-CoA.5
The growth of yeasts on fatty acids as carbon source is accompanied by proliferation of peroxisomes, in which β-oxidation is exclusively housed in these organisms. Unlike in mammalian peroxisomes, the properties of peroxisomal enzymes in yeast allow β-oxidation to go to completion. Two out of the four enzymatic activities of β-oxidation reside in the same protein in one polypeptide chain. These are the 2-enoyl-CoA hydratase and the 3-hydroxyacyl-CoA dehydrogenase activities of multifunctional enzymes type 1 and 2 (MFE-1 and MFE-2, respectively), the difference being that type 1 is specific for the (S)-isomeric substrates and type 2 for the (R)-isomers. Despite functional similarity, MFE-1 and -2 are structurally very different,6 as is MFE-2 from mammals and yeasts. The 3-hydroxyacyl-CoA dehydrogenase domain in yeast MFE-2 has interesting features; namely it has undergone a duplication (Figure 1)7 and evolved into two (3R)-hydroxyacyl-CoA dehydrogenases in the same polypeptide with different chain-length specificities.8 The first dehydrogenase (dehydrogenase A; the amino-terminal region of the sequence in MFE-2) can catalyze the reaction for medium- and long-chain substrates ((3R)-OH-C10–(3R)-OH-C16), whereas the second dehydrogenase (dehydrogenase B; the region of the sequence between dehydrogenase A and hydratase 2 in MFE-2) shows the highest activity with short-chain substrates ((3R)-OH-C4). The substrate specificities of the dehydrogenase A and B domains have been exploited in yeast for the synthesis of polyhydroxyalkanoates (PHA), a polyester with biodegradable plastic properties, made from the polymerization of 3-hydroxyacyl-CoA intermediates.9 Expression of variants of the dehydrogenase with inactivation of either the A or B domain along with a peroxisomal PHA synthase leads to the synthesis of PHA with a different monomer composition, which ultimately influences polymer properties.
The mechanism of substrate preference by yeast dual dehydrogenases is unknown. Here, we have studied the substrate binding modes of Candida tropicalis dehydrogenase A and B, which are short-chain alcohol dehydrogenase/reductase (SDR) superfamily members, by first determining the crystal structure of MFE-2 truncated for the hydratase 2 unit (MFE-2h2Δ), but containing the dehydrogenase AB heterodimer, followed by docking studies with two different chain-length substrates. Based on identification of the key structural determinants of substrate binding to dehydrogenases and the results from kinetic measurements, we propose the structural basis for the observed substrate specificity difference of the (3R)-hydroxyacyl-CoA dehydrogenases in C. tropicalis MFE-2.
Section snippets
Structure determination
Multiple amino acid sequence alignment (Figure 2) reveals that stretches of non-conserved amino acid residues separate (3R)-hydroxyacyl-CoA dehydrogenase A and B as well as the dehydrogenase B and 2-enoyl-CoA hydratase 2 units in C. tropicalis peroxisomal MFE-2. Using the second stretch as a guide to separate (3R)-hydroxyacyl-CoA dehydrogenases from 2-enoyl-CoA hydratase 2, and aiming to get a homogenous protein suited for structural studies, four different variants were tested, resulting in the
Discussion
Although the structures of C. tropicalis and rat (3R)-hydroxyacyl-CoA dehydrogenases are overall very similar, certain features in the structures give rise to important differences in the substrate specificities of these enzymes. In all cases, the binding of the CoA part is transmitted by a similar hydrogen bonding pattern, whereas the binding of the acyl groups shows remarkable differences.
Concerning dehydrogenase A, which is active with long and medium-chain substrates (kcat with C16:C10:C4
Construction of the plasmid for overexpression
The MFE-2h2Δ construction used for this study contains the two dehydrogenase units but lacks the hydratase 2 unit from the C. tropicalis MFE-2. The construction was modified from the original clone8 and cloned into pET3a vector for expression in E. coli BL21 Codon Plus (DE3)-RIL cells as described.10 The construction contains amino acid residues 1–604, a C-terminal hexahistidine tail and includes point mutations Thr506Ser and Phe508Met.
Protein production, purification and crystallization
These methods have been described.10 Briefly: induction of
Acknowledgements
This work was supported by the FP6 European Union Project “Peroxisomes” (LSHG-CT-2004-512018) and by the Université de Lausanne and by grants from the Academy of Finland (no. 201262 and no. 211109), the Sigrid Juselius Foundation and the Fonds National Suisse de la Recherche Scientifique (3100A0-105874). We thank Dr Kristian Koski and Dr Tomi Airenne for practical and theoretical help during structure determination; Marika Kamps, Eeva-Liisa Stefanius, and Ville Ratas for technical assistance;
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