Crystal structure and biochemical characterization of beta-keto thiolase B from polyhydroxyalkanoate-producing bacterium Ralstonia eutropha H16

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Highlights

Abstract

ReBktB is a β-keto thiolase from Ralstonia eutropha H16 that catalyzes condensation reactions between acetyl-CoA with acyl-CoA molecules that contains different numbers of carbon atoms, such as acetyl-CoA, propionyl-CoA, and butyryl-CoA, to produce valuable bioproducts, such as polyhydroxybutyrate, polyhydroxybutyrate-hydroxyvalerate, and hexanoate. We solved a crystal structure of ReBktB at 2.3 Å, and the overall structure has a similar fold to that of type II biosynthetic thiolases, such as PhbA from Zoogloea ramigera (ZrPhbA). The superposition of this structure with that of ZrPhbA complexed with CoA revealed the residues that comprise the catalytic and substrate binding sites of ReBktB. The catalytic site of ReBktB contains three conserved residues, Cys90, His350, and Cys380, which may function as a covalent nucleophile, a general base, and second nucleophile, respectively. For substrate binding, ReBktB stabilized the ADP moiety of CoA in a distinct way compared to ZrPhbA with His219, Arg221, and Asp228 residues, whereas the stabilization of β-mercaptoethyamine and pantothenic acid moieties of CoA was quite similar between these two enzymes. Kinetic study of ReBktB revealed that Km, Vmax, and Kcat values of 11.58 μM, 1.5 μmol/min, and 102.18 s−1, respectively, and the catalytic and substrate binding sites of ReBktB were further confirmed by site-directed mutagenesis experiments.

Introduction

Ralstonia eutropha H16 is a gram-negative lithoautotrophic bacterium that inhabits soil and freshwater [1]. R. eutropha has received a significant amount of attention from the biotechnology community because it can utilize both organic compounds and molecular hydrogen (H2) as energy sources. Furthermore, R. eutropha can synthesize polyhydroxyalkanoates (PHA) polymers while storing surplus organic compounds [2], [3]. Recently, the analysis of the R. eutropha genome revealed genes involved in the biosynthesis of PHA [2], [3], and the granule shaped carbon polymer synthesized by R. eutropha has been extensively used to make biodegradable thermoplastics [4], [5], [6].

Among many different types of PHAs, R. eutropha mainly biosynthesizes the polyhydroxybutyrate (PHB) monopolymer [7] by utilizing three enzymes, β-ketothiolase (PhbA), NADPH-dependent acetoacetyl-CoA reductase (PhbB), and PHB synthase (PhbC), whose coding genes are located on the same operon [8], [9], [10], [11]. β-ketothiolase is an enzyme that catalyzes the first step of PHA synthesis, and is also involved in many other important biosynthetic pathways [12], [13]. Thiolases can be divided two categories, type I degradative (EC 2.3.1.16) and type II biosynthetic (EC 2.3.1.9) thiolases. Among the 37 β-ketothiolase homologues that are present in the R. eutropha genome, two β-ketothiolases, PhbA and β-ketothiolase B (BktB), are known to play a role in the biosynthesis of PHA by catalyzing Claisen condensation reactions of 2 molecules of acetyl-CoA to form acetoacetyl-CoA [14].

Although the functions of RePhbA and ReBktB are similar as β-ketothiolase enzymes, ReBktB is also involved in the biosynthesis of longer chain polymers in R. eutropha. ReBktB catalyzes not only a condensation reaction between 2 acetyl-CoA molecules to produce acetoacetyl-CoA, but it also catalyzes a condensation reaction between acetyl-CoA and propionyl-CoA to produce valeryl-CoA. On the other hand, RePhbA utilizes acetyl-CoA as its sole substrate and produces acetoacetyl-CoA [7]. Due to the function of ReBktB, this enzyme has been used in the synthesis of poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBHV) or longer chain copolymers [7]. Furthermore, ReBktB has been shown to catalyze a condensation reaction between acetyl-CoA and butyryl-CoA to form 3-ketohexanoyl-CoA, which can be used to produce hexanoate or n-hexanol [15].

In the present study, we report a crystal structure of β-ketothiolase B from R. eutropha H16 (ReBktB) and reveal its residues involved in substrate binding. Biochemical properties of ReBktB were also elucidated by kinetic analysis and site-directed mutagenesis experiments. Importantly, our results provide useful information for engineering ReBktB to have an increased rate of producing valuable bio-products such as bio-plastics and bio-fuels.

Section snippets

Cloning, expression, and purification

Cloning, expression, purification, and crystallization of ReBktB will be described elsewhere (Kim et al., in preparation). Briefly, the recombinant ReBktB protein was expressed using the pPROEX Hta (Invitrogen) bacterial expression system and purified through sequential chromatographic steps including Ni–NTA, ion-exchange, and size-exclusion chromatography. All purification experiments were performed at 4 °C. The degree of protein purification was confirmed by SDS–PAGE. The purified protein was

Overall structure of ReBktB

ReBktB is an enzyme that catalyzes a condensation reaction between acetyl-CoA with acyl-CoA molecules with a different number of carbon atoms, such as acetyl-CoA, propionyl-CoA, and butyryl-CoA, to produce acetoacetyl-CoA, valeryl-CoA, and 3-ketohexanoyl-CoA, respectively. The enzymatic products are further converted to valuable bioproducts, such as PHB, PHBHV, and hexanoate. To investigate the structural basis for the catalytic mechanism of β-ketothiolase from R. eutropha H16 (ReBktB), we

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MEST) (NRF-2009-C1AAA001-2009-0093483) and by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the MEST (ABC- 2012-053895), and also funded by a part of the project titled “Gyeongbuk Sea Grant Program” funded by the MLTM, Korea.

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These two authors contributed equally to this work.

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