Crystal structure and biochemical properties of the (S)-3-hydroxybutyryl-CoA dehydrogenase PaaH1 from Ralstonia eutropha

https://doi.org/10.1016/j.bbrc.2014.04.101Get rights and content

Highlights

  • We determined a crystal structure of 3-hydroxy-butyryl-CoA reductase from Ralstonia eutropha H16 (RePaaH1).

  • Structures in complex with NAD+ and acetoacetyl-CoA revealed cofactor and substrate binding modes of the enzyme.

  • Kinetic and biochemical properties of RePaaH1 were revealed.

Abstract

3-Hydroxybutyryl-CoA dehydrogenase is an enzyme involved in the synthesis of the biofuel n-butanol by converting acetoacetyl-CoA to 3-hydroxybutyryl-CoA. To investigate the molecular mechanism of n-butanol biosynthesis, we determined crystal structures of the Ralstonia eutropha-derived 3-hydroxybutyryl-CoA dehydrogenase (RePaaH1) in complex with either its cofactor NAD+ or its substrate acetoacetyl-CoA. While the biologically active structure is dimeric, the monomer of RePaaH1 comprises two separated domains with an N-terminal Rossmann fold and a C-terminal helical bundle for dimerization. In this study, we show that the cofactor-binding site is located on the Rossmann fold and is surrounded by five loops and one helix. The binding mode of the acetoacetyl-CoA substrate was found to be that the adenosine diphosphate moiety is not highly stabilized compared with the remainder of the molecule. Residues involved in catalysis and substrate binding were further confirmed by site-directed mutagenesis experiments, and kinetic properties of RePaaH1were examined as well. Our findings contribute to the understanding of 3-hydroxybutyryl-CoA dehydrogenase catalysis, and will be useful in enhancing the efficiency of n-butanol biosynthesis by structure based protein engineering.

Introduction

Due to issues such as limited fossil fuel availability, greenhouse gas emissions, and the requirement for increased energy security or diversity, there is increased public and scientific interest in energy alternatives such as biofuels. A wide range of biofuels can be derived from plant or microbial biomass [1]. The two major biofuels in use today are ethanol and butanol, which can be combined with gasoline for use in conventional engines [2], [3]. However, ethanol has a low energy efficiency compared to gasoline and high vaporizability [4]. Alternatively, n-butanol produced by microbial fermentation has characteristics that are closer to those of motor-vehicle fuels and could serve as a better replacement [5]. The anaerobic bacterium Clostridium acetobutylicum can efficiently produce n-butanol through a carbohydrate catabolic pathway [6], [7]. In comparison with bio-ethanol, the advantage of the biosynthesized n-butanol is that it has a high energy content, low corrosion, increased solubility, and easier to blend with gasoline [8], [9], [10].

Even if n-butanol is considered a potential next generation biofuel source, its biosynthetic efficiency must be improved, and there have been multiple attempts to do so [11]. For example, many engineering efforts ranging from genetic modifications to microbial culture optimization, have aimed to increase n-butanol production during ABE fermentation. However, the n-butanol synthetic titers do not exceed 1 g/L in heterologous host cells that express clostridial n-butanol biosynthetic machinery [7], [8], [9], [10]. Very recently, alternative methods to enhance the n-butanol yield have been reported; these involve the use of metabolically engineered hosts such as Escherichia coli, Pseudomonas putida, and Bacillus subtilis in the n-butanol biosynthetic pathway to improve biofuel production from small organic molecules [12], [13], [14].

A next step to produce large amount of n-butanol is the engineering of non-solventogenic microbes [15]. It has been shown that the n-butanol inhibits E. coli growth for example, the growth is almost ceased at approximately n-butanol concentrations of 1% [16], therefore, the toxicity effects of n-butanol in bacterial cells should be moderated [17]. Another issue is that the additive pathways for n-butanol synthesis disrupt the balance of energy carriers such as NADH/NAD+, which results in a decrease in n-butanol production [18]. These multiple issues have necessitated an optimization of the heterologous metabolic pathways to maximize the n-butanol biosynthetic yield by the use of engineered non-solventogenic microbes [18], [19].

In contrast to C. acetobutylicum, which is a representative n-butanol producing host, Ralstonia eutropha has a broader spectrum and is used in the production of polymers such as polyhydroxybutyrate (PHB). It has been reported that the 3-hydroxybutyryl-CoA dehydrogenase PaaH1 is involved in n-butanol biosynthesis [20], and R. eutropha-derived PaaH1 is proposed as a homolog of Clostridium butyricum 3-hydroxybutyryl-CoA dehydrogenase (CbHBD) that is involved in the second step of n-butanol biosynthesis [21]. Here, we report the first crystal structure of R. eutropha 3-hydroxybutyryl-CoA dehydrogenase (RePaaH1), an enzyme that catalyzes the second step of n-butanol biosynthesis and converts acetoacetyl-CoA to 3-hydroxybutyryl-CoA. Kinetic properties and mutagenesis experiments were also reported.

Section snippets

Preparation of RePaaH1

Cloning, expression, purification, and crystallization of RePaaH1 will be described elsewhere. Briefly, the RePaaH1 coding gene (Met1-Lys284, M.W. 32 kDa) was amplified by polymerase chain reaction (PCR) using R. eutropha chromosomal DNA as a template. The PCR product was then subcloned into pET30a (Invitrogen) with 6-histag at the C-terminus. The expression construct was transformed into an E. coli B834 strain, which was grown in 1 L of LB medium containing kanamycin (50 mg/ml) at 37 °C. After

Overall structure of RePaaH1

To determine enzymatic properties of the RePaaH1 protein, we determined the crystal structure of RePaah1 at 2.6 Å. The asymmetric unit contains three RePaaH1 molecules, which corresponded to one biologically active dimer and one molecule that can be generated to a dimer by crystallographic symmetry operation (Fig. 1). The size exclusion chromatography results also confirmed that RePaaH1 exists as a dimer (data not shown). A search using the Dali server revealed that the structure of RePaaH1 was

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), Korea.

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    These authors contributed equally to this work.

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