Structure and biochemical studies of a pseudomonad maleylpyruvate isomerase from Pseudomonas aeruginosa PAO1
Introduction
The opportunistic human pathogen Pseudomonas aeruginosa PAO1 is a model organism of the Pseudomonas genus that is famous for having versatile features such as environmental ubiquitousness [1], quorum sensing [2], and biofilm formation [3]. Non-actinomycete species such as P. aeruginosa are also known for metabolic diversity and can be found in a variety of habitats. P. aeruginosa can utilize various aromatic hydrocarbons as a carbon source such as toluene [4], naphthalene [5], phenethylamines [6], fluoranthene [7], and pyrene [8]. It has been shown that rhamnolipids, which are well-known biosurfactants produced from P. aeruginosa, positively effect on the biodegradation of oils and aromatic substances by increasing hydrocarbon mobility [9,10]. Thus, there is interest in the use of these pseudomonads in bioremediation for the removal of aromatic pollutants in the environment.
Gentisate is one of the most important foothold chemicals in the degradation pathway of salicylate [11], xylenol [12], naphthalene [13], and anthracene [14]. The key enzymes involved in the aerobic gentisate catabolism are gentisate dioxygenase (GDO), which breaks the aromaticity of benzenoid through the introduction of oxygen; maleylpyruvate isomerase (MPI), which isomerizes cis-form maleylpyruvate to trans-form fumarylpyruvate; and fumarylpyruvate hydrolase (FPH), which hydrolyzes the β-ketone moiety and produces the central metabolites pyruvate and fumarate (Fig. 1A). Among them, MPI is reported to be glutathione- [15], mycothiol- [16], and cysteine-dependent [17].
An operon containing genes for the gentisate pathway exists in P. aeruginosa (PA2470, PaGDO; PA2471, PaFPH; and PA2473, PaMPI). PaMPI is predicted to belong to the ζ-class of the glutathione S-transferase superfamily, which has poor glutathione conjugation activity against common substrates. Maleylacetoacetate isomerase (MaaI) is a well-known member of this class, and it is a penultimate enzyme counterpart of MPI in the eukaryotic degradation pathways of the aromatic amino acid [18]. Relatively, there is not much research on bacterial MPI involved in aromatic hydrocarbon degradation, and the detailed comparison of the MPI structures have not been performed. In this study, we aimed to characterize the pseudomonad MPI by determining its crystal structure. The derived crystal structure and comparison with other glutathione S-transferases suggest putative substrate-accommodating residues, most of which are exclusively harbored by PaMPI. In addition, we elucidated the biochemical function and properties of PaMPI.
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Protein preparation
The PaMPI coding gene was amplified through polymerase chain reaction (PCR) and the PCR products were sub-cloned into pET30a (Novagen) expression vector. The pET30a- PaMPI was transformed into an Escherichia coli BL21 (DE3)-T1R strain and was grown in 1 L of LB medium containing kanamycin (50 mg L-1) at 37 °C to OD600 of 0.6. After induction with 1.0 mM Isopropyl 1-thio-b-d-galactopyranoside (IPTG) for further 20 h at 18 °C, the culture medium was harvested by centrifugation at 4000 rpm for
Overall structure of PaMPI
To provide the molecular structure of PaMPI, we determined its crystal structure at a 1.86 Å resolution (Table 1). The asymmetric unit contains two molecules forming a dimer, which represents the physiological oligomeric form of PaMPI. Residues Met1-Pro30 and Leu52-Tyr211 in molecule I and Met1-Val31 and Ala 51-Leu212 in molecule II were visible in the electron density map and modeled. The DALI analysis [27] showed that the structural homologues of PaMPI are MaaI from Anaeromyxobacter
Acknowledgements
This work was supported by the Technology Development Program to Solve Climate Changes of the National Research Foundation funded by the Ministry of Science and Innovation, New Zealand (NRF-2017M1A2A2087631). H. Seo is supported by the Global PhD Fellowship Program of the Korean Government (2018H1A2A1061751).
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These authors contributed equally to this work.