Structural and molecular dynamic studies of Pseudomonas aeruginosa OdaA reveal the regulation role of a C-terminal hinge element

https://doi.org/10.1016/j.bbagen.2020.129756Get rights and content

Highlights

  • Crystal structures of OdaA in apo-form and CoA-bound form are solved.

  • OdaA has different binding affinities for CoA derivatives.

  • Dynamics simulations using the crystallographic structures were performed.

  • Dynamics of the C-terminal region of OdaA is crucial for substrate coupling.

Abstract

Background

Crotonase superfamily members exhibit great catalytic diversity towards various acyl-CoA substrates. A common CoA moiety binding pattern is usually observed in this family, understanding the substrate-binding mechanism would facilitate the rational engineering of crotonases for improved properties.

Methods

We applied X-ray crystallography to investigate a putative enoyl-CoA hydratase/isomerase OdaA in Pseudomonas aeruginosa. Thermal shift assay (TSA) were performed to explore the binding of OdaA with CoA thioester substrates. Furthermore, we performed molecular dynamics (MD) simulations to elucidate the dynamics of its CoA-binding site.

Results

We solved the crystal structures of the apo and CoA-bound OdaA. Thermal shift assay (TSA) showed that CoA thioester substrates bind to OdaA with a different degree. MD simulations demonstrated that the C-terminal alpha helix underwent a structural transition and a hinge region would associate with this conformational change.

Conclusions

TSA in combination with MD simulations elucidate that the dynamics of C-terminal alpha helix in CoA-binding, and a hinge region play an important role in conformational change.

General significance

Those results help to extend our knowledge about the nature of crotonases and would be informative for future mechanistic studies and industry applications.

Introduction

Crotonase superfamily (CS) enzymes catalyze a broad spectrum of chemical reactions, including (de)hydration, isomerization, dehalogenation, (de)carboxylation, formation or cleavage of carbon‑carbon bonds, and thioester hydrolysis [1,2]. The catalytic versatility of CS enzymes enable them to play multiple physiological and pathological roles from prokaryote to eukaryote [[3], [4], [5], [6]]. Thus, some CS members are considered to be potential targets for cancer therapy or antimicrobial treatments [2,[7], [8], [9]]. Furthermore, CS-catalyzed reactions exhibit considerable diversity in their ability to catalyze ring-structures, carbon‑carbon and carbon-hetero atom bonds, providing unique opportunities to engineer novel biocatalysts for industrial applications [10].

All CS members recruit a conserved overall architecture termed as crotonase fold, where the repeated ββα units arrange into two almost perpendicular sections [1]. Most CS-catalyzed reactions are featured by the stabilization of an enolate/oxyanion intermediate via two backbone NH groups in an oxyanion hole, commonly with substrates activated as coenzyme A (CoA) thioesters [1,10]. The diverse reaction mechanisms of different CS enzymes are accomplished by integrating various properties and arrangements of active site residues or/and distinct substrate binding pockets [11,12]. It is worthy to note that most CS enzymes interact with the substrates via its C-terminal tail [2,13], therefore,to investigate the conformational states that account for its dynamic transitions is a central issue in understanding the activity regulation of those enzymes. In addition, computer-aided methods [14] are playing an important role in protein structure and design, and molecular dynamics simulation technology is used to explore the details of the interaction between proteins and small molecules [15].

In this study, we solved the three-dimensional structure of OdaA, a putative enoyl-coenzyme A (CoA) hydratase/isomerase (ECH/ECI) OdaA from Pseudomonas aeruginosa [4]. Structural analysis suggests that OdaA functions as a trimer and has a ECI active site which catalyzes double-bond isomerization reaction. The thermal shift assay (TSA) experiments demonstrate that the C-terminal segment is crucial in acyl-CoA substrate anchoring and molecular dynamic (MD) simulation analysis reveals that the conformational transition of C-terminal alpha helix is responsible for substrate binding regulation. Notably, residue Arg232 and Glu237 located the hinge of this alpha helix are important during the conformational transition. These results extend the regulatory mechanisms of CS enzymes and provide information for future engineering strategies.

Section snippets

Protein expression and purification

The full-length odaA gene (PA4330) was amplified from the P. aeruginosa PAO1 genomic DNA by polymerase chain reaction (PCR) using gene-specific primers. The coding region of odaA was inserted into plasmid pET-22b (+) containing six C-terminal histidine residues (LEHHHHHH) using ClonExpressTM II One Step Cloning Kit (Vazyme Biotech Co, Ltd., Nanjing). The pET-22b-odaA was transformed into E. coli strain BL21 (DE3) for protein expression. The bacterial culture was grown in Luria–Bertani (LB)

OdaA structure exhibits a conserved crotonase fold with a isomerase active site

To understand the structural characteristics of OdaA, we constructed a recombinant OdaA-pET22b expression vector and purified the protein to homogeneity. Samples were subjected to crystallization trials with or without CoA, X-ray diffraction data were collected and solved to 1.9 Å for both apo-form and CoA-bound form. Most of the residues have been built in the electron density map except the flexible C-terminal tail (residues Arg249-Ala257). The detailed crystallographic and refinement

Discussion

In bacteria, the cis-trans isomerization of unsaturated fatty acids have been recognized as crucial mechanisms in many important aspects of rapid responses to environmental stress, cellular signaling and pathogenesis [39,40]. For instance, a new group of quorum sensing (QS) signal molecule, named as diffusible signal factors (DSFs), is a type of cis-2-unsaturated fatty acids and function to control biofilm formation, antibiotic resistances and virulence factor production [3,41,42]. The DSF

Conclusion

To date, the oligomeric nature of most crotonases and the conformational changes are very important for ECH/ECI catalysis. Hence, the knowledge of crotonases structures, in particular the ligand-bound complex, will be informative for exploring the substrate binding mechanism. Our study via x-ray structural, along with MD simulation results propose instructive information about the regulation mechanism of OdaA and could contribute to understanding more details of the CS enzyme catalytic property

Accession numbers

Atomic coordinates of the refined structures have been deposited in the Protein Data Bank (www.pdb.org) with the PDB code 7CRD (OdaA) and 7BOR (CoA-bound OdaA).

Credit author statement

Ning-lin Zhao: Designed the research, performed the related experiment, analyzed the data and wrote the manuscript.

Qian-qian Zhang: Designed the research, performed the dynamic simulations, analyzed the data and wrote the manuscript.

Chang Zhao and Li Liu: Analyzed the data.

Tao Li, Chang-cheng Li and Li-hui He: Directed the experiment.

Yi-bo Zhu and Ying-jie Song: Directed the experiment.

Huan-xiang Liu and Rui Bao: Analyzed the data, wrote the manuscript and edited.

Declaration of Competing Interest

The authors have no competing interests.

Acknowledgements

The work was financially supported by National Key Research and Development Plan under Grants [grant number 2016YFA0502700]; National Natural Science Foundation of China [grant numbers 81871615, 81670008]; National Mega-project for Innovative Drugs [grant number 2019ZX09721001-001-001]; Ministry of Science and Technology of the People's Republic of China [grant number 2018ZX09201018-005]; Luzhou Municipal People's Government-Southwest Medical University Science and Technology Strategic

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