Biochemical and Biophysical Research Communications
Crystal structure of an acetyl-CoA acetyltransferase from PHB producing bacterium Bacillus cereus ATCC 14579
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)
- et al.
Structural insights into polyhydroxyalkanoates biosynthesis
Trends Biochem. Sci.
(2018) - et al.
The chemomechanical properties of microbial polyhydroxyalkanoates
Prog. Polym. Sci.
(2014) - et al.
Polyhydroxyalkanoate inclusion-body growth and proliferation in Bacillus megaterium
FEMS Microbiol. Lett.
(1996) - et al.
Biosynthesis and structural characterization of polyhydroxyalkanoates produced by Pseudomonas aeruginosa ATCC 27853 from long odd-chain fatty acids
Int. J. Biol. Macromol.
(2018) - et al.
Class I and III polyhydroxyalkanoate synthases from Ralstonia eutropha and Allochromatium vinosum: characterization and substrate specificity studies
Arch. Biochem. Biophys.
(2001) - et al.
Processing of X-ray diffraction data collected in oscillation mode
Methods Enzymol.
(1997) - et al.
Microplastics as an emerging threat to terrestrial ecosystems
Global Change Biol.
(2018) - et al.
Microplastics: an introduction to environmental transport processes
Wires Water
(2018) - et al.
Accumulation and fragmentation of plastic debris in global environments
Philos. Trans. R. Soc. Lond. B Biol. Sci.
(2009) - et al.
Our plastic age
Philos. T R Soc. B
(2009)
Application of (R)-3-hydroxyalkanoate methyl esters derived from microbial polyhydroxyalkanoates as novel biofuels
Biomacromolecules
A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry
Chem. Soc. Rev.
Bacterial polymers: biosynthesis, modifications and applications
Nat. Rev. Microbiol.
Cited by (10)
Production and characterization of biodegradable polymer-polyhydroxybutyrate from agricultural waste-sugarcane bagasse by the novel marine bacterium Klebsiella pneumoniae G1
2022, Bioresource Technology ReportsCitation Excerpt :PHA type, PHB has a lot of applications in food packaging and biomedical applications (Wei et al., 2011). The PHB synthesis involves a repertoire of enzymes, thiolase (phaA), reductase (phaB), and synthase (phaC) required to convert acetyl coenzyme A (precursor) to PHB (Hong et al., 2020). The strains of Bacillus, Pseudomonas, Ralstonia, and Alcaligenes are the native PHB producers (McAdam et al., 2020).
p-Nitrophenyl esters provide new insights and applications for the thiolase enzyme OleA
2021, Computational and Structural Biotechnology JournalCitation Excerpt :OleA is a homodimer in the thiolase superfamily of enzymes and catalyzes a non-decarboxylative Claisen condensation of two acyl-CoA substrates consisting of C8-C16 carbon chains to produce hydrocarbons and natural products [17] (Fig. 1A). Thiolase enzymes are involved in many different pathways in the metabolism of fatty acids, polyhydroxybutyrate storage, and natural product biosynthesis [18–22]. OleA homologs that produce natural products include NltAB, a heterodimer biosynthesized by a Nocardia species that produces the β-lactone nocardiolactone [23] (Fig. 1, inset).
Antimicrobial Activity of Eugenol Against Bacillus cereus and Its Application in Skim Milk
2024, Foodborne Pathogens and DiseaseHarnessing economical biopolymer extrusion: the Bacillus clade as endotoxin-free platforms for next-generation bioprocesses
2024, Reviews in Environmental Science and BiotechnologyApplication of PHA surface binding proteins of alkali-tolerant Bacillus as surfactants
2024, Brazilian Journal of Microbiology
- 1
These authors contributed equally to this work.