Crystal structure and biochemical characterization of beta-keto thiolase B from polyhydroxyalkanoate-producing bacterium Ralstonia eutropha H16 Eun-Jung Kim a,1 , Hyeoncheol Francis Son a,1 , Sangwoo Kim a,b , Jae-Woo Ahn a , Kyung-Jin Kim a,⇑ a Structural and Molecular Biology Laboratory, School of Life Sciences and Biotechnology, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, Republic of Korea b School of Nono-Bioscience and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798, Republic of Korea article info Article history: Received 1 January 2014 Available online 22 January 2014 Keywords: Beta-keto thiolase Ralstonia eutropha Crystal structure Polyhydroxyalkanoates abstract ReBktB is a b-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 b-mercaptoethyamine and pantothenic acid moieties of CoA was quite similar between these two enzymes. Kinetic study of ReBktB revealed that K m , V max , and K cat values of 11.58 lM, 1.5 lmol/min, and 102.18 s À1 , respectively, and the catalytic and substrate binding sites of ReBktB were further confirmed by site-directed mutagenesis experiments. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction Ralstonia eutropha H16 is a gram-negative lithoautotrophic bac- terium that inhabits soil and freshwater [1]. R. eutropha has re- ceived a significant amount of attention from the biotechnology community because it can utilize both organic compounds and molecular hydrogen (H 2 ) as energy sources. Furthermore, R. eutro- pha 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 [4–6]. Among many different types of PHAs, R. eutropha mainly bio- synthesizes the polyhydroxybutyrate (PHB) monopolymer [7] by utilizing three enzymes, b-ketothiolase (PhbA), NADPH-dependent acetoacetyl-CoA reductase (PhbB), and PHB synthase (PhbC), whose coding genes are located on the same operon [8–11]. b-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 b-ketothiolase homologues that are present in the R. eutropha genome, two b-ketothiolases, PhbA and b-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 b- 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 pro- duce 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(b-hydroxybuty- rate-co-b-hydroxyvalerate) (PHBHV) or longer chain copolymers [7]. Furthermore, ReBktB has been shown to catalyze a condensa- tion reaction between acetyl-CoA and butyryl-CoA to form 3-keto- hexanoyl-CoA, which can be used to produce hexanoate or n-hexanol [15]. 0006-291X/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2014.01.055 ⇑ Corresponding author. Fax: +82 53 955 5522. E-mail address: kkim@knu.ac.kr (K.-J. Kim). 1 These two authors contributed equally to this work. Biochemical and Biophysical Research Communications 444 (2014) 365–369 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc