Biochemical and Biophysical Research Communications
Crystal structure and biochemical characterization of beta-keto thiolase B from polyhydroxyalkanoate-producing bacterium Ralstonia eutropha H16
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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2020, Metabolic EngineeringCitation Excerpt :In general, all seven thiolase enzymes conferred the ability to consume octanoic acid and produce 2-nonanone (Figs. 5B and S5A). In terms of ranking, we found strains expressing ReBktB (Kim et al., 2014) and SeFadA exhibited the highest consumption rates (over 80% octanoic acid consumed within 18 h) and 2-nonanone titers (~280 mg/L) (Figs. 5B and S5B). Next, we evaluated 2-nonanone production from glycerol in the background strain ΔfadRAI pTRC99a-FadD6-PsFadM + pACYC-ReBktB + pBTRCK-CpFatB1*.
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2018, Trends in Biochemical SciencesCitation Excerpt :However, the detailed molecular mechanisms of PHA biosynthesis have remained unclear due to the lack of structural information on PHA biosynthetic enzymes. Recently, several research groups have reported determination of the crystal structures of the three key enzymes, PhaA, PhaB, and PhaC, of C. necator H16 [30–35]. These recent studies presenting structural and biochemical characteristics of the key enzymes are instrumental in understanding the molecular mechanisms of PHA biosynthesis.
Combination of type II fatty acid biosynthesis enzymes and thiolases supports a functional β-oxidation reversal
2018, Metabolic EngineeringCitation Excerpt :To investigate the use of FAB enzymes in this context, a thiolase with longer chain length specificity was utilized in place of AtoB. Specifically, the 3-ketoacyl-CoA thiolase encoded by bktB from Ralstonia eutropha (Kim et al., 2014) was selected given its ability to support the synthesis of C6-C10 acyl-CoAs and corresponding carboxylic acids (Clomburg et al., 2015; Kim et al., 2015). Utilizing a previously developed chromosomal bktB expression construct in place of atoB (Clomburg et al., 2015), the expression of this thiolase in conjunction with FabG, FabZ, and FabI resulted in the production of carboxylic acids up to C10 (Fig. 4A).
Coenzyme A-free activity, crystal structure, and rational engineering of a promiscuous β-ketoacyl thiolase from Ralstonia eutropha
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2015, Metabolic EngineeringCitation Excerpt :Considering our success with a chromosomal-based, cumate-controlled expression system for individual components of the β-oxidation reversal pathway at native E. coli atoB and fadB chromosomal sites (Vick et al., 2015), this system was selected for the expression of all core/elongation enzymes required for the functional β-oxidation reversal. Due to the potential limitations with the chain length specificity of AtoB or FadA as the thiolase component for C6–C10 carboxylic acid production, the 3-ketoacyl-CoA thiolase encoded by bktB from R. eutropha (Kim et al., 2014) was selected as a potential alternative due in part to previous reports indicating the ability of this enzyme to condense acetyl-CoA with C4 and C6 acyl-CoA intermediates (Machado et al., 2012; Martin et al., 2013). To provide chromosomal expression of bktB, a cat-sacB cassette was constructed at the atoB locus enabling seamless replacement of atoB with bktB while retaining cumate-controlled expression (see Section 2).
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These two authors contributed equally to this work.