Dataset Information

Critical Functions of Region 1-67 and Helix XIII in Retaining the Active Structure of NhaD Antiporter in Halomonas sp. Y2.

ABSTRACT: NhaD-type antiporters are mainly distributed in various Proteobacteria, especially in marine microorganisms and human pathogens. This distribution as well as the pathogenic properties of these strains suggest that these antiporters contribute to the regulation of high osmoregulation and are potential drug targets. Two NhaD homologs, NhaD1 and NhaD2, from the halotolerant and alkaliphilic Halomonas sp. Y2 exhibits similar, high in vitro activity, but remarkably different in vivo functions. To search for critical domains or residues involved in these differences of physiological functions, various chimeras composed of NhaD1 and NhaD2 segments were generated. Two regions at residues 1-67 and 464-492 were found to be responsible for the robust in vivo function of NhaD2, and region 464-492 is also crucial to the pH response of the antiporter. In particular, the completely abolished activity of KNabc/N463r, highly recovered activity while very weakly recovered ion resistance of the KNabc/N463r-C7 chimera, suggested that transmembrane helix (TM) XIII is crucial for the robust ion resistance of NhaD2. Using site-directed mutagenesis, seven hydrophobic residues in TM XIII were identified as key residues for the ion translocation of NhaD2. Compared with the fluorescence resonance energy transfer (FRET) profile in the wild-type NhaD2, the reduced FRET efficiency of N463r chimeras provided solid evidence for conformational changes in the N463r fusion protein and consequently verified the structural functions of TM XIII in the pH activation and physiological functions of NhaD2.

SUBMITTER: Yang Z

PROVIDER: S-EPMC5942162 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Critical Functions of Region 1-67 and Helix XIII in Retaining the Active Structure of NhaD Antiporter in Halomonas sp. Y2.

Yang Zhou Z Meng Yiwei Y Zhao Qi Q Cheng Bin B Xu Ping P Yang Chunyu C

Frontiers in microbiology 20180502

NhaD-type antiporters are mainly distributed in various Proteobacteria, especially in marine microorganisms and human pathogens. This distribution as well as the pathogenic properties of these strains suggest that these antiporters contribute to the regulation of high osmoregulation and are potential drug targets. Two NhaD homologs, NhaD1 and NhaD2, from the halotolerant and alkaliphilic Halomonas sp. Y2 exhibits similar, high in vitro activity, but remarkably different i ...[more]

PMID: 29770128

Similar Datasets

Project description:Two NhaD-type antiporters, NhaD1 and NhaD2, from the halotolerant and alkaliphilic Halomonas sp. strain Y2, exhibit different physiological functions in regard to Na+ and Li+ resistance, although they share high sequence identity. In the present study, the truncation of an additional 4 C-terminal residues from NhaD2 or an exchange of 39 N-terminal residues between these proteins resulted in the complete loss of antiporter activity. Interestingly, combining 39 N-terminal residues and 7 C-terminal residues of NhaD2 (N39D2-C7) partially recovered the activity for Na+ and Li+ expulsion, as well as complementary growth following exposure to 300 mM Na+ and 100 mM Li+ stress. The recovered activity of chimera N39D2-C7 indicated that the N and C termini are structurally dependent on each other and function synergistically. Furthermore, fluorescence resonance energy transfer (FRET) analysis suggested that the N and C termini are relatively close in proximity which may account for their synergistic function in ion translocation. In the N-terminal region of N39D2-C7, the replacement of Glu38 with Pro abolished the recovered complementary and transport activities. In addition, this amino acid substitution in NhaD2 resulted in a drastically decreased complementation ability in Escherichia coli KNabc (level identical to that of NhaD1), as well as decreased activity and an altered pH profile.IMPORTANCE Limited information on NhaD antiporters supports speculation that these antiporters are important for resistance to high salinity and alkalinity. Moreover, only a few functional residues have been identified in NhaD antiporters, and there is limited literature on the molecular mechanisms of NhaD antiporter activity. The altered antiporter abilities of chimeras and mutants in this study implicate the functions of the N and C termini, especially Glu38, in pH regulation and ion translocation, and, most importantly, the essential roles of this negatively charged residue in maintaining the physiological function of NhaD2. These findings further our understanding of the molecular mechanism of NhaD antiporters for ion transport.

Project description:BACKGROUND:As an attracted compatible solute, 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) showed great potentials in various field. However, lower productivity and high saline medium seriously hinder its wide applications. RESULTS:The entire ectoine metabolism, including pathways for ectoine synthesis and catabolism, was identified in the genome of an ectoine-excreting strain Halomonas hydrothermalis Y2. By in-frame deletion of genes encoding ectoine hydroxylase (EctD) and (or) ectoine hydrolase (DoeA) that responsible for ectoine catabolism, the pathways for ectoine utilization were disrupted and resulted in an obviously enhanced productivity. Using an optimized medium containing 100 g L-1 NaCl in a 500-mL flask, the double mutant of Y2/?ectD/?doeA synthesized 3.13 g L-1 ectoine after 30 h cultivation. This is much higher than that of the wild type strain (1.91 g L-1), and also exceeds the production of Y2/?ectD (2.21 g L-1). The remarkably enhanced accumulation of ectoine by Y2/?ectD/?doeA implied a critical function of Doe pathway in the ectoine catabolism. Furthermore, to reduce the salinity of fermentation medium and overcome the wastewater treatment difficulty, mutants that lacking key Na+/H+ antiporter, Mrp and (or) NhaD2, were constructed based on strain Y2/?ectD/?doeA. As a result, the Mrp-deficient strain could synthesize equal amount of ectoine (around 7 g L-1 or 500 mg (g DCW) -1) in the medium containing lower concentration of NaCl. During a fed-batch fermentation process with 60 g L-1 NaCl stress, a maximum 10.5 g L-1 ectoine was accumulated by the Mrp-deficient strain, with a specific production of 765 mg (g DCW)-1 and a yield of 0.21 g g-1 monosodium glutamate. CONCLUSION:The remarkably enhanced production of ectoine by Y2/?ectD/?doeA implied the critical function of Doe pathway in the ectoine catabolism. Moreover, the reduced salinity requirement of Mrp-deficient strain implied a feasible protocol for many compatible solute biosynthesis, i.e., by silencing some Na+/H+ antiporters in their halophilic producers and thus lowering the medium salinity.

Dataset Information

Critical Functions of Region 1-67 and Helix XIII in Retaining the Active Structure of NhaD Antiporter in Halomonas sp. Y2.

Publications

Critical Functions of Region 1-67 and Helix XIII in Retaining the Active Structure of NhaD Antiporter in <i>Halomonas</i> sp. Y2.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets