Crystal structure and biochemical properties of ReH16_A1887, the 3-ketoacyl-CoA thiolase from Ralstonia eutropha H16

https://doi.org/10.1016/j.bbrc.2015.02.148Get rights and content

Highlights

  • We determined a crystal structure of ReH16_A1887, a 3-ketoacyl-CoA thiolase from Ralstonia eutropha.

  • Structural change of the lid-subdomain upon the binding of the substrate was observed in ReH16_A1887.

  • Binding mode of CoA substrate in ReH16_A1887 was determined.

Abstract

ReH16_A1887 from Ralstonia eutropha is an enzyme annotated as a 3-ketoacyl-CoA thiolase, and it catalyzes the fourth step of β-oxidation degradative pathways by converting 3-ketoacyl-CoA to acyl-CoA. We determined the crystal structures of ReH16_A1887 in the apo-form and in complex with its CoA substrate. ReH16_A1887 functions as a dimer, and the monomer of ReH16_A1887 comprises three subdomains (I, II, and III). The structural comparison between the apo-form and the CoA-bound form revealed that ReH16_A1887 undergoes a structural change in the lid-subdomain (subdomain III) upon the binding of the CoA substrate. The CoA molecule was stabilized by hydrogen bonding with positively charged residues such as Lys18, Arg210, and Arg217, and residues Thr213 and Gln151 aid its binding as well. At the active site of ReH16_A1887, highly conserved residues such as Cys91, His348, and Cys378 were located near the thiol-group of CoA, indicating that ReH16_A1887 might catalyze the thiolase reaction in a way similar to other thiolases. Moreover, in the vicinity of the covalent nucleophile Cys91, a hydrophobic hole that might serve as a binding site for the acyl-group of 3-ketoacyl-CoA was observed. The residues involved in enzyme catalysis and substrate-binding were further confirmed by site-directed mutagenesis experiments.

Introduction

Ralstonia eutropha H16 first attracted biotechnological interest nearly 50 years ago with the realization that its ability to produce and store large amounts of polyhydroxyalkanoate (PHA) that could be harnessed to make biodegradable plastics. The strain can store PHA up to 80% of its cell dry weight as a result of nutrient limitation [1]. Many groups have explored production of PHAs from renewable carbon sources such as plant oils. Plant oils are a suitable carbon source for this endeavor as 3-hydroxyacyl coenzyme A (3-hydroxyacyl-CoA) PHA precursors can be produced from intermediates in the fatty acid degradation pathway [2], [3]. Plant oils consist of triacylglycerols (TAGs), in which three fatty acids are joined to a glycerol backbone. Recently, plant oils have been explored as a possible feedstock alternative to petroleum for chemical production [4]. These oils can also be used as sources of carbon for bioplastic production by bacteria such as R. eutropha. R. eutropha must therefore employ a fatty acid degradation pathway to consume oils and fatty acids.

Fatty acids are fundamental biomolecules that are abundant in all life forms. With their enormous variation in chain length and degree of saturation, they are essential for energy storage, form structural entities in biomembranes, and serve as signaling molecules. Fatty acids are broken down in a cyclic manner, two carbons at a time, to generate a range of products by the process known as β-oxidation [5]. The shortened fatty acyl-CoA can then be subjected to further rounds of β-oxidation or directed to other pathways. The fatty acid β-oxidation spiral involves four enzymes, acyl-CoA dehydrogenase (ACD), 2-enoyl-CoA hydratase (ECH), L-3-hydroxyacyl-CoA dehydrogenase (HACD) and 3-ketoacyl-CoA thiolase (KACT) [6]. Among these enzymes, KACT catalyzes the degradative cleavage of a β-ketoacyl-CoA to acyl-CoA and a two-carbon shortened acyl-CoA [7], [8].

There are two distinct forms of 3-ketoacyl-CoA thiolases. Type I is the 3-ketoacyl-CoA thiolase (EC 2.3.1.16), a catabolic enzyme performing the reverse Claisen condensation reaction involved in such as the β-oxidation cycle. Type II is the acetoacetyl-CoA thiolase (ACAT; EC 2.3.1.9), which is involved in the anabolic mevalonate pathway performing Claisen condensation. R. eutropha possesses both type I and II thiolases. The fatty acid β-oxidation pathway in R. eutropha is uncharacterized in the literature. Most studies of microbial fatty acid β-oxidation have been conducted in Escherichia coli and Bacillus subtilis [9], [10], although some information is available regarding fatty acid degradation in Pseudomonas species [11], [12]. A search of the R. eutropha H16 genome reveals many potential β-oxidation pathway gene homologs [13]. For example, 50 genes in the R. eutropha H16 genome are annotated as enoyl-CoA hydratases and 46 genes are annotated as acyl-CoA dehydrogenases. However, it is not known which of these homologs actually play a role in fatty acid breakdown.

In this study, we aimed to determine the crystal structure of Ralstonia eutropha 3-ketoacyl-CoA thiolase A1887 (ReH16_A1887), an enzyme that catalyzes the fourth step of β-oxidation degradative pathways and converts 3-ketoacyl-CoA to acyl-CoA. Biochemical and mutagenesis experiments were also performed.

Section snippets

Preparation of H16_A1887

Cloning, expression, purification, and crystallization of ReH16_A1887 will be described elsewhere (Kim et al., in preparation). Briefly, the ReH16_A1887 coding gene (Met1-Leu392, M.W. 41.5 kDa) was amplified by polymerase chain reaction using R. eutropha chromosomal DNA as a template. The PCR product was then subcloned into pET30a (Invitrogen), and the resulting expression vector pET30a: ReH16_A1887 was transformed into an E. coli BL21(DE3)-T1R strain, which was grown in 1 L of LB medium

Overall structure of ReH16_A1887

To elucidate the enzymatic properties of the ReH16_A1887 protein, we determined the crystal structure of ReH16_A1887 at 1.4 Å. The asymmetric unit contains two ReH16_A1887 molecules, which corresponded to one biologically active dimer (Fig. 1). The size exclusion chromatography results also confirmed that ReH16_A1887 exists as a dimer (data not shown). A search using the Dali server revealed that the structure of ReH16_A1887 was homologous to that of FadA5, a thiolase from M. tuberculosis (Mt

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MEST) (2014R1A2A2A01005752 and NRF-2014M1A2A2033626) and by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the MEST (2012M3A6A2053895).

References (22)

  • D. Knoop

    Industrial Conciliation and Arbitration

    (1905)
  • Cited by (5)

    View full text