Characterization And Mutation Analysis Of Two NhaD Type Na~+/H~+ Antiporters From Halomonas Sp. Y2

As important second Na+ extrusion system,Na+/H+ antiporters(NHAs)play very important role in maintaining cytoplasmic Na+(Li+)and pH homeostasis of bacteria.They can extrude Na+(Li+)out of cells through transmembranc proton electrochemical gradient engendered by primary proton pumps,and simultaneously uptake H+ into cells.By this way to maintain cytoplasmic Na+(Li+)at a low level.Because of strong Na+ extrusion systems,moderately halophilic bacteria can survive under a wide range of extracellular NaCl concentrations.In our previous study,a moderately halophilic Halomonas sp.Y2 was isolated from alkaline paper-making black liquor.Four different Na+/H+ antiporters encoding genes were found from its genome sequence.Among them,two NhaD type antiporters,designating as NhaDl?NhaD2 were cloned into Na+/H+ deficient strain E.coli KNabc.NCBI BLAST reveals that NahD1 and NhaD2 exhibited 72%identity in amino acid sequence.Besides,these two antiporters also had more than 50%identity in amino acid sequence with some erported NhaD antiporters,like He-NhaD,Aa-NhaD,Vp-NhaD and Vc-NhaD.Firstly,we investigated the complementation of NhaD1 and NhaD2 to deficient strain E.coli KNabc.As a result,NhaDl could only restore the E.coli KNabc grow under 200 mM NaCl or 100 mM LiCl,no obvious complementation was observed under diffenence pHs.However,NhaD2 exhibited strong complementation ability to make E.coli KNabc grow in the presence of 500 mM NaCl or 400 mM LiCl,and under stress can restore E.coli KNabc grow under pH 8.0(Na+)and pH 8.5(Li+).EMVs assay reveals they both exhibited strong Na+/H+ and Li+/H+ antiport activities at alkaline pH,despite of the great difference of NhaD1 and NhaD2 in complementary growth.Besides,they share the same Li+/H+ antiport pH profile(highest activity observed at pH 9.5)and apparent Km for Na+ and Li+(Na+ was a better substrate than Li+).Interesting,NhaD1 and NhaD2 showed different pH sensitivity conferring Na+transporting.NhaD1 was active at pH 6.0 and most active at pH 8.0-8.5 whereas NhaD2 lacked activity at pH 6.0 and had the pH optima at pH 9.5 or higher.Secondly,based on sequence comparison,11 mutants were constructed in this study for identifying important sites on its transport properties,especially on pH sensitivity.As a result,V86I,N166D,M168L,M172L,I236L,N247S and I346V mutants completely abolished the Na+/H+ and Li+/H+ antiport activities at all pHs,indicated that these sites are indispensable for NhaD1.A87G?S200A and S353A retained decreased activities for Na+ and Li+ transporting and exhibited similar pH profiles with wild type NhaDl.S282A exhibited alkaline shift pH profile in compared with the wild type of NhaD1 with about 1.5 pH alkaline shift of its pH optima.This site was predicted locating in TMS VIII and supposed to be important in the protein's pH sensitivity,combined with some other nearby residues.Besides,S353A significantly decreased binding affnnity of Na+ and Li+ with a 10-fold increase of apparent Km for Na+ and Li+.At last,based on their topology model,a series of NhaD1 and NhaD2 chimeric antiporters were constructed and their Na+/H+ and Li+/H+ antiport activities were measured.Sequence alignment indicated NhaD1 and NhaD2 share low identity between 1-39 regions.We suppose that they had different and special structure at these regions and their structure were irreplaceable.C-terminal of NhaD2(489SVYG492)plays very important role in maintain antiport activities.The C-terminal of NhaD2(463-492)region is indispensable and may take part in pH regulation.C463rD2-C7 chimeric antiporters only can store 300 mM NaC1(OD600nm?0.2),with a large difference with wide type NhaD2.This indicate 463-485 area of NhaD1 paly important roles in complementary growth.