Crystal structure and biochemical properties of the (S)-3- hydroxybutyryl-CoA dehydrogenase PaaH1 from Ralstonia eutropha Jieun Kim a,1 , Jeong Ho Chang b,1 , Kyung-Jin Kim a,⇑ a School of Life Sciences, KNU Creative BioResearch Group (BK21 Plus Program), Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, Republic of Korea b Department of Biology, Teachers College, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, Republic of Korea article info Article history: Received 3 April 2014 Available online 29 April 2014 Keywords: (S)-3-Hydroxybutyryl-CoA dehydrogenase Ralstonia eutropha n-Butanol Crystal structure 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-hydroxybu- tyryl-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 aden- osine 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 exper- iments, and kinetic properties of RePaaH1were examined as well. Our findings contribute to the under- standing of 3-hydroxybutyryl-CoA dehydrogenase catalysis, and will be useful in enhancing the efficiency of n-butanol biosynthesis by structure based protein engineering. Ó 2014 Elsevier Inc. All rights reserved. 1. 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 bio- fuels in use today are ethanol and butanol, which can be combined with gasoline for use in conventional engines [2,3]. However, eth- anol 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 biosynthe- sized n-butanol is that it has a high energy content, low corrosion, increased solubility, and easier to blend with gasoline [8–10]. Even if n-butanol is considered a potential next generation bio- fuel 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 micro- bial culture optimization, have aimed to increase n-butanol pro- duction 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 [7–10]. 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 [12–14]. A next step to produce large amount of n-butanol is the engi- neering 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 path- ways for n-butanol synthesis disrupt the balance of energy carriers such as NADH/NAD + , which results in a decrease in n-butanol http://dx.doi.org/10.1016/j.bbrc.2014.04.101 0006-291X/Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding author. Address: Structural and Molecular Biology Laboratory, School of Life Sciences, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, Republic of Korea. Fax: +82 53 955 5522. E-mail address: kkim@knu.ac.kr (K.-J. Kim). 1 These authors contributed equally to this work. Biochemical and Biophysical Research Communications 448 (2014) 163–168 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc