Crystal structure of an acetyl-CoA acetyltransferase from PHB producing bacterium Bacillus cereus ATCC 14579

https://doi.org/10.1016/j.bbrc.2020.09.048Get rights and content

Highlights

  • We determined the crystal structures of thiolase from Bacillus cereus (BcTHL) in an apo-form and the CoA-complexed form.

  • We identified the residues involved in the enzyme catalysis and the substrate binding mode of BcTHL.

  • We revealed that the Arg221 residue undergoes a positional conformational change upon the binding of the CoA molecule.

Abstract

Bacillus cereus ATCC 14579 is a known polyhydroxybutyrate (PHB)-producing microorganism that possesses genes associated with PHB synthesis such as PhaA, PhaB, and PHA synthases. PhaA (i.e., thiolase) is the first enzyme in the PHA biosynthetic pathway, which catalyze the condensation of two acetyl-CoA molecules to acetoacetyl-CoA. Our study elucidated the crystal structure of PhaA in Bacillus cereus ATCC 14579 (BcTHL) in its apo- and CoA-bound forms. BcTHL adopts a type II biosynthetic thiolase structure by forming a tetramer. The crystal structure of CoA-complexed BcTHL revealed that the substrate binding site of BcTHL is constituted by different residues compared with other known thiolases. Our study also revealed that Arg221, a residue involved in ADP binding, undergoes a positional conformational change upon the binding of the CoA molecule.

Introduction

Plastics are the most widely used polymers and have become an essential part of our daily lives. However, they also cause severe problems on environment and human health. Thus, over the past decades, intensive efforts have been made to develop eco-friendly and biodegradable plastics [[1], [2], [3], [4]]. Since polyhydroxyalkanoates (PHAs) have chemical properties similar to the conventional plastics, they can be natural, sustainable, and biodegradable polymers and used to produce bioplastics as a substitute for petroleum-based plastics [[5], [6], [7]]. PHAs can also be used in various applications such as packaging materials, agriculture, and the food industry [8,9]. In numerous microorganisms, PHAs are synthesized and stored in the cell cytoplasm as water-insoluble inclusions under nutrient-limiting conditions [10,11]. Microorganisms, such as Cupriavidus necator [12], Pseudomonas sp. [13], and Bacillus sp. [14,15], have been extensively studied on biosynthesis of PHAs, and all of these organisms possess genes involved in the PHA biosynthetic pathway.

Three key enzymes, such as PhaA, PhaB, and PhaC, are involved in the PHA biosynthetic pathway, and these three enzymes are also known as acetyl-CoA acetyltransferase, acetoacetyl-CoA reductase, and PHA synthase, respectively (Fig. 1A) [16]. First, PhaA catalyzes the condensation reaction of two acetyl-CoA molecules into acetoacetyl-CoA, and second, PhaB converts acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA. Finally, PhaC catalyzes the polymerization of the (R)-3-hydroxybutyryl-unit to synthesize the polymer. The PHA synthesis process can be classified into 4 types depending on the genetic arrangement of PHA synthase [17]. Class I and II PHA synthases are encoded by the phaC gene in C. necator and phaC1 and phaC2 genes in Pseudomonas aeruginosa, respectively. The class III PHA synthase has two subunits encoded by phaC and phaE, which have been reported in Allochromatium vinosum. Class IV PHA synthase is encoded by the phaC and phaE genes found in Bacillus megaterium [[18], [19], [20]].

PhaA, an enzyme involved in the first reaction of the PHA pathway, belongs to the type II biosynthetic thiolases. The enzyme utilizes two cysteines and one histidine for enzyme catalysis and the reaction mechanism of the enzyme is divided into two steps, a covalent catalysis step and a condensation step [21]. In the covalent catalysis step, the thiol group of one cysteine attacks a thiol ester bond of the acetyl-CoA substrate, resulting in the formation of the acetyl-S-enzyme intermediate. In the condensation step, the other cysteine deprotonates the hydroxyl-group of the second acetyl-CoA substrate, which enables the second substrate attack the acetyl-S-enzyme intermediate and the formation of acetoacetyl-CoA.

Bacillus cereus ATCC 14579 (B. cereus ATCC 14579) is a Gram-positive, facultative anaerobic, spore-producing, motile, and rod-shaped bacterium [22]. The strain is also known to produce polyhydroxybutyrate (PHB), a type of PHA, and possesses genes associated with the synthesis of PHB [14,15]. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG), the B. cereus ATCC 14579 strain possesses three phaA candidates in its genome, including BC3627, BC4023, and BC5344. However, biochemical properties of these enzymes have not been characterized yet. Therefore, our study sought to elucidate the crystal structure of thiolase (i.e., the product of the BC5344 gene) from B. cereus ATCC 14579 (BcTHL). Based on the CoA-complexed structure of thiolase and site-directed mutagenesis experiments, we also elucidated the substrate binding mode of this enzyme.

Section snippets

Preparation of BcTHL proteins

The BcTHL coding gene was amplified from chromosomal DNA of B. cereus ATCC 14579 by polymerase chain reaction (PCR), and subcloned into pET30a expression vector. The E. coli BL21 (DE3)-T1R strain was used for the protein expression host. The cells transformed with the pET30a:BcTHL vector were grown to an OD600 of 0.6 in a Luria-Bertani medium supplemented with 100 mg L−1 kanamycin at 37 °C. The BcTHL protein expression was induced by adding 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG)

Overall structure of BcTHL

To elucidate the enzymatic properties of the BcTHL protein, we determined the crystal structure of the enzyme at 1.56 Å (Fig. 2A). The refined structure was in good agreement with the X-ray crystallographic statistics of bond angles, bond length, and other geometric parameters (Supplementary Table 1). Our crystallographic data demonstrate that the overall structure of BcTHL is similar to those of type II biosynthetic thiolases, such as the thiolase of Clostridium acetobutylicum (CaTHL, PDB code

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the Cooperative Research Program for Agricultural Science & Technology Development (project no. PJ01492602), Rural Development Administration, Republic of Korea.

References (27)

  • X. Zhang et al.

    Application of (R)-3-hydroxyalkanoate methyl esters derived from microbial polyhydroxyalkanoates as novel biofuels

    Biomacromolecules

    (2009)
  • G.Q. Chen

    A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry

    Chem. Soc. Rev.

    (2009)
  • B.H. Rehm

    Bacterial polymers: biosynthesis, modifications and applications

    Nat. Rev. Microbiol.

    (2010)
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