Effects of Specific Amino Acid Substitutions on Activities of Dinitrogenase Reductase-Activating Glycohydrolase fromRhodospirillum rubrum

ABSTRACT Site-directed mutagenesis of the draG gene was used to generate altered forms of dinitrogenase reductase-activating glycohydrolase (DRAG) with D123A, H142L, H158N, D243G, and E279R substitutions. The amino acid residues H142 and E279 are not required either for the coordination to the metal center or for catalysis since the variants H142L and E279R retained both catalytic and electron paramagnetic resonance spectral properties similar to those of the wild-type enzyme. Since DRAG-H158N and DRAG-D243G variants lost their ability to bind Mn(II) and to catalyze the hydrolysis of the substrate, H158 and D243 residues could be involved in the coordination of the binuclear Mn(II) center in DRAG.

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Site-directed mutagenesis of the putative active site of human 17β-hydroxysteroid dehydrogenase type 1

Biochemical Journal ◽

10.1042/bj3040289 ◽

1994 ◽

Vol 304 (1) ◽

pp. 289-293 ◽

Cited By ~ 52

Author(s):

T J Puranen ◽

M H Poutanen ◽

H E Peltoketo ◽

P T Vihko ◽

R K Vihko

Keyword(s):

Amino Acid ◽

Catalytic Properties ◽

Catalytic Efficiency ◽

Hydroxysteroid Dehydrogenase ◽

Site Directed Mutagenesis ◽

Dimensional Structure ◽

Amino Acid Residues ◽

Wild Type ◽

Wild Type Enzyme

Several amino acid residues (Cys54, Tyr155, His210, His213 and His221) at a putative catalytic site of human 17 beta-hydroxysteroid dehydrogenase type 1 were mutated to Ala. Replacement of His221 by Ala remarkably reduced the catalytic activity, which resulted from a change of both the Km and the Vmax. values of the enzyme. Compared with the wild-type enzyme, the catalytic efficiency of the His221-->Ala mutant was reduced 20-fold for the oxidative reaction and 11-fold for the reductive reaction. With similar mutations at His210 or His213, no notable effects on the catalytic properties of the enzyme were detected. However, a simultaneous mutation of these amino acid residues decreased the Vmax. values of both oxidation and reduction by about 50% from those measured for the wild-type enzyme. Although Cys54 has been localized in the cofactor-binding region of the enzyme, a Cys54-->Ala mutation did not lead to changes in the enzymic activity. The most dramatic effects on the catalytic properties of the enzyme were achieved by mutating Tyr155, which resulted in an almost completely inactivation of the enzyme. The decreased enzymic activities of the Tyr155-->Ala, His210-->Ala + His213-->Ala and His221-->Ala mutations were also reflected in a reduced immunoreactivity of the enzymes. The results thus suggest that the lower catalytic efficiency of the mutant enzymes is due to an exchange of catalytically important amino acid residues and/or remarkable alterations in the three-dimensional structure of the enzyme. The recently detected polymorphisms (Ala237<-->Val and Ser312<-->Gly) were not found to affect either the catalytic or the immunological properties of the type 1 enzyme.

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Is there a "dolichol recognition sequence" in enzymes that interact with dolichols and other polyisoprenoid substrates?

Acta Biochimica Polonica ◽

10.18388/abp.1994_4715 ◽

1994 ◽

Vol 41 (3) ◽

pp. 269-274 ◽

Cited By ~ 2

Author(s):

J S Schutzbach

Keyword(s):

Amino Acid ◽

Deletion Mutant ◽

Site Directed Mutagenesis ◽

Amino Acid Residues ◽

Lipid Matrix ◽

Wild Type ◽

Wild Type Enzyme ◽

Recognition Sequence ◽

Apparent Affinity ◽

Conserved Domain

Yeast dolichyl-P-mannose synthase and a number of other enzymes that interact with dolichol or dolichyl-P as substrates contain a highly conserved amino-acid sequence that has been proposed as a potential dolichol recognition sequence [Albright, C.F., Orlean, P. & Robbins, P.W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7366-7369]. In dolichyl-P-mannose synthase, the most highly conserved amino-acid residues of this domain were modified by site directed mutagenesis, and for one construct the sequence was completely deleted. Enzymes containing the site directed modifications, and the deletion mutant, were found to retain catalytic activity, and all of the modified enzymes had the same apparent affinity for Dol-P as wild type enzyme when assayed in a phospholipid matrix. Based on these results, the amino-acid composition and sequence of the conserved domain are not critically important for the recognition and binding of Dol-P when the synthase is reconstituted in a lipid matrix.

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Expression of recombinant human serum amyloid A in mammalian cells and demonstration of the region necessary for high-density lipoprotein binding and amyloid fibril formation by site-directed mutagenesis

Biochemical Journal ◽

10.1042/bj3181041 ◽

1996 ◽

Vol 318 (3) ◽

pp. 1041-1049 ◽

Cited By ~ 48

Author(s):

Himakshi PATEL ◽

Jo BRAMALL ◽

Helen WATERS ◽

Maria C. DE BEER ◽

Patricia WOO

Keyword(s):

Amino Acid ◽

Serum Amyloid A ◽

Amyloid Fibrils ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Site Directed Mutagenesis ◽

Amino Acid Residues ◽

Wild Type ◽

Amyloid A ◽

Amyloid Fibril Formation

Site-directed mutagenesis of the acute-phase human serum amyloid A (SAA1α) protein was used to evaluate the importance of the N-terminal amino acid residues, namely RSFFSFLGEAF. The full-length cDNA clone of SAA1α (pA1.mod.) was used to create two mutations, namely Gly-8 to Asp-8 and an 11 amino acid truncation between Arg-1 and Phe-11 respectively. Wild-type and mutant cDNAs were expressed in Chinese hamster ovary (CHO) cells under the control of the human cytomegalovirus promoter, which resulted in the secretion of the processed proteins into the culture media. Wild-type recombinant human SAA (rSAA) protein was shown to have pI values of 6.0 and 6.4, similar to the human SAA isoform SAA1α and SAA1α desArg found in acute-phase plasma. N-terminal sequencing of 56 residues confirmed its identity with human SAA1α. The total yield of wild-type rSAA measured by ELISA was between 3.5 and 30 mg/l. The two mutations resulted in reduced expression levels of the mutant SAA proteins (3–10 mg/l). Further measurements of rSAA concentration in lipid fractions of culture medium collected at a density of 1.21 g/ml (high-density lipoprotein; HDL) and 1.063–1.18 g/ml (very-low-density lipoprotein/low-density lipoprotein; VLDL/LDL) showed that 76% of the wild-type protein was found in the HDL fraction and the remaining 24% in the infranatant non-lipid fraction. In contrast the relative concentration of mutant rSAA in HDL and infranatant fractions was reversed. This is consistent with the previously proposed involvement of the 11 amino acid peptide in anchoring SAA protein on to HDL3 [Turnell, Sarra, Glover, Baum, Caspi, Baltz and Pepys (1986) Mol. Biol. Med.3, 387–407]. Wild-type rSAA protein was shown to form amyloid fibrils in vitro under acidic conditions as shown by electron microscopy, and stained positive with Congo Red and exhibited apple-green birefringence when viewed under polarized light. Under the same conditions mutSAA(G8D) and mutSAAΔ1–11 did not form amyloid fibrils. In conclusion, replacement of Gly-8 by Asp-8 or deletion of the first 11 amino acid residues at the N-terminus of rSAA diminishes its capacity to bind to HDL and decreases amyloid fibril formation.

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Structure-based identification of inositol polyphosphate 1-phosphatase fromEntamoeba histolytica

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714021245 ◽

2014 ◽

Vol 70 (11) ◽

pp. 3023-3033 ◽

Cited By ~ 2

Author(s):

Khaja Faisal Tarique ◽

Syed Arif Abdul Rehman ◽

Christian Betzel ◽

Samudrala Gourinath

Keyword(s):

Amino Acid ◽

Metal Binding ◽

Three Dimensional ◽

Protozoan Parasite ◽

Amino Acid Residues ◽

Inositol Polyphosphate ◽

Inositol Monophosphatase ◽

Specific Amino Acid ◽

Fructose 1,6 Bisphosphate ◽

Hydrolysis Of

Inositol polyphosphate 1-phosphatase fromEntamoeba histolytica(EhIPPase) is an Mg2+-dependent and Li+-sensitive enzyme that catalyzes the hydrolysis of inositol 1,4-bisphosphate [Ins(1,4)P2] intomyo-inositol 1-monophosphate and PO43−. In the present work, EhIPPase has been biochemically identified and its crystal structure has been determined in the presence of Mg2+and PO43−at 2.5 Å resolution. This enzyme was previously classified as a 3′(2′),5′-bisphosphate nucleotidase in the NCBI, but its biochemical activity and structural analysis suggest that this enzyme behaves more like an inositol polyphosphate 1-phosphatase. The ability of EhIPPase to hydrolyze the smaller Ins(1,4)P2 better than the bulkier 3′-phosphoadenosine 5′-phosphate (PAP) is explained on the basis of the orientations of amino-acid residues in the binding site. This structure is the first of its class to be determined from any protozoan parasite, and is the third to determined among all organisms, following its rat and bovine homologues. The three-dimensional fold of EhIPPase is similar to those of other members of the inositol monophosphatase superfamily, which also includes inositol monophosphatase, 3′(2′),5′-bisphosphate nucleotidase and fructose-1,6-bisphosphate 1-phosphatase. They all share conserved residues essential for metal binding and substrate hydrolysis, with the motif D-Xn-EE-Xn-DP(I/L)DG(S/T)-Xn-WD-Xn-GG. The structure is divided into two domains, namely α+β and α/β, and the substrate and metal ions bind between them. However, the ability of each enzyme class to act specifically on its cognate substrate is governed by the class-specific amino-acid residues at the active site.

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Molecular and Functional Analyses of Kunjin Virus Infectious cDNA Clones Demonstrate the Essential Roles for NS2A in Virus Assembly and for a Nonconservative Residue in NS3 in RNA Replication

Journal of Virology ◽

10.1128/jvi.77.14.7804-7813.2003 ◽

2003 ◽

Vol 77 (14) ◽

pp. 7804-7813 ◽

Cited By ~ 123

Author(s):

Wen Jun Liu ◽

Hua Bo Chen ◽

Alexander A. Khromykh

Keyword(s):

Amino Acid ◽

Virus Assembly ◽

Nonstructural Protein ◽

Rna Replication ◽

Site Directed Mutagenesis ◽

Single Amino Acid ◽

Cdna Clones ◽

Amino Acid Residues ◽

Wild Type ◽

Common Amino Acid

ABSTRACT A number of full-length cDNA clones of Kunjin virus (KUN) were previously prepared; it was shown that two of them, pAKUN and FLSDX, differed in specific infectivities of corresponding in vitro transcribed RNAs by ∼100,000-fold (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). In this study, we analyzed a possible genetic determinant(s) of the observed differences in infectivity initially by sequencing the entire cDNAs of both clones and comparing them with the published sequence of the parental KUN strain MRM61C. We found six common amino acid residues in both cDNA clones that were different from those in the published MRM61C sequence but were similar to those in the published sequences of other flaviviruses from the same subgroup. pAKUN clone had four additional codon changes, i.e., Ile59 to Asn and Arg175 to Lys in NS2A and Tyr518 to His and Ser557 to Pro in NS3. Three of these substitutions except the previously shown marker mutation, Arg175 to Lys in NS2A, reverted to the wild-type sequence in the virus eventually recovered from pAKUN RNA-transfected BHK cells, demonstrating the functional importance of these residues in viral replication and/or viral assembly. Exchange of corresponding DNA fragments between pAKUN and FLSDX clones and site-directed mutagenesis revealed that the Tyr518-to-His mutation in NS3 was responsible for an ∼5-fold decrease in specific infectivity of transcribed RNA, while the Ile59-to-Asn mutation in NS2A completely blocked virus production. Correction of the Asn59 in pAKUN NS2A to the wild-type Ile residue resulted in complete restoration of RNA infectivity. Replication of KUN replicon RNA with an Ile59-to-Asn substitution in NS2A and with a Ser557-to-Pro substitution in NS3 was not affected, while the Tyr518-to-His substitution in NS3 led to severe inhibition of RNA replication. The impaired function of the mutated NS2A in production of infectious virus was complemented in trans by the helper wild-type NS2A produced from the KUN replicon RNA. However, replicon RNA with mutated NS2A could not be packaged in trans by the KUN structural proteins. The data demonstrated essential roles for the KUN nonstructural protein NS2A in virus assembly and for NS3 in RNA replication and identified specific single-amino-acid residues involved in these functions.

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Alteration of Chain Length Substrate Specificity of Aeromonas caviae R-Enantiomer-Specific Enoyl-Coenzyme A Hydratase through Site-Directed Mutagenesis

Applied and Environmental Microbiology ◽

10.1128/aem.69.8.4830-4836.2003 ◽

2003 ◽

Vol 69 (8) ◽

pp. 4830-4836 ◽

Cited By ~ 31

Author(s):

Takeharu Tsuge ◽

Tamao Hisano ◽

Seiichi Taguchi ◽

Yoshiharu Doi

Keyword(s):

Amino Acid ◽

Substrate Specificity ◽

Chain Length ◽

Acyl Chain ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Acyl Chain Length ◽

Aeromonas Caviae

ABSTRACT Aeromonas caviae R-specific enoyl-coenzyme A (enoyl-CoA) hydratase (PhaJAc) is capable of providing (R)-3-hydroxyacyl-CoA with a chain length of four to six carbon atoms from the fatty acid β-oxidation pathway for polyhydroxyalkanoate (PHA) synthesis. In this study, amino acid substitutions were introduced into PhaJAc by site-directed mutagenesis to investigate the feasibility of altering the specificity for the acyl chain length of the substrate. A crystallographic structure analysis of PhaJAc revealed that Ser-62, Leu-65, and Val-130 define the width and depth of the acyl-chain-binding pocket. Accordingly, we targeted these three residues for amino acid substitution. Nine single-mutation enzymes and two double-mutation enzymes were generated, and their hydratase activities were assayed in vitro by using trans-2-octenoyl-CoA (C8) as a substrate. Three of these mutant enzymes, L65A, L65G, and V130G, exhibited significantly high activities toward octenoyl-CoA than the wild-type enzyme exhibited. PHA formation from dodecanoate (C12) was examined by using the mutated PhaJAc as a monomer supplier in recombinant Escherichia coli LS5218 harboring a PHA synthase gene from Pseudomonas sp. strain 61-3 (phaC1 Ps). When L65A, L65G, or V130G was used individually, increased molar fractions of 3-hydroxyoctanoate (C8) and 3-hydroxydecanoate (C10) units were incorporated into PHA. These results revealed that Leu-65 and Val-130 affect the acyl chain length substrate specificity. Furthermore, comparative kinetic analyses of the wild-type enzyme and the L65A and V130G mutants were performed, and the mechanisms underlying changes in substrate specificity are discussed.

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Expression and mutagenesis of the catalytic domain of cGMP-inhibited phosphodiesterase (PDE3) cloned from human platelets

Biochemical Journal ◽

10.1042/bj3230217 ◽

1997 ◽

Vol 323 (1) ◽

pp. 217-224 ◽

Cited By ~ 19

Author(s):

K. Mary TANG ◽

Elliott K. JANG ◽

Richard J. HASLAM

Keyword(s):

Amino Acid ◽

Catalytic Domain ◽

Site Directed Mutagenesis ◽

Deletion Mutants ◽

Amino Acid Residues ◽

Degenerate Primers ◽

Hydrolytic Activities ◽

N Terminus ◽

Hydrolysis Of ◽

Camp And Cgmp

We have used reverse transcriptase PCR, platelet mRNA and degenerate primers based on platelet peptide sequences, to amplify a fragment of platelet cGMP-inhibited phosphodiesterase (cGI-PDE; PDE3). Sequence analysis of this clone established that both the platelet and the cardiac forms of PDE3 were derived from the same gene (PDE3A). A RT-PCR product representing the C-terminal half of platelet PDE3 cDNA and corresponding to amino acid residues 560-1141 of the cardiac enzyme, was cloned and expressed in Escherichia coli cGI-PDEΔ1. Further deletion mutants were constructed by removing either an additional 100 amino acids from the N-terminus (cGI-PDEΔ2) or the 44-amino-acid insert characteristic of the PDE3 family, from the catalytic domain (cGI-PDEΔ1Δi). In addition, site-directed mutagenesis was performed to explore the function of the 44-amino-acid insert. All mutants were evaluated for their ability to hydrolyse cAMP and cGMP, their ability to be photolabelled by [32P]cGMP and for the effects of PDE3 inhibitors. The Km values for hydrolysis of cAMP and cGMP by immunoprecipitates of cGI-PDEΔ1 (182±12 nM and 153±12 nM respectively) and cGI-PDEΔ2 (131±17 nM and 99±1 nM respectively) were significantly lower than those for immunoprecipitates of intact platelet PDE3 (398±50 nM and 252±16 nM respectively). Moreover, N-terminal truncations of platelet enzyme increased the ratio of Vmax for cGMP/Vmax for cAMP from 0.16±0.01 in intact platelet enzyme, to 0.37±0.05 in cGI-PDEΔ1 and to 0.49±0.04 in cGI-PDEΔ2. Thus deletion of the N-terminus enhanced hydrolysis of cGMP relative to cAMP, suggesting that N-terminal sequences may exert selective effects on enzyme activity. Removal of the 44-amino-acid insert generated a mutant with a catalytic domain closely resembling those of other PDE gene families but despite a limited ability to be photolabelled by [32P]cGMP, no cyclic nucleotide hydrolytic activities of the mutant were detectable. Mutation of amino acid residues in putative β-turns at the beginning and end of the 44-amino-acid insert to alanine residues markedly reduced the ability of the enzyme to hydrolyse cyclic nucleotides. The PDE3 inhibitor, lixazinone, retained the ability to inhibit cAMP hydrolysis and [32P]cGMP binding by the N-terminal deletion mutants and the site-directed mutants, suggesting that PDE3 inhibitors may interact exclusively with the catalytic domain of the enzyme.

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Site-directed mutagenesis and substrate-induced inactivation of β-lactamase I

Biochemical Journal ◽

10.1042/bj2881045 ◽

1992 ◽

Vol 288 (3) ◽

pp. 1045-1051 ◽

Cited By ~ 7

Author(s):

S J Thornewell ◽

S G Waley

Keyword(s):

Alpha Helix ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Beta Lactamase ◽

Wild Type ◽

Wild Type Enzyme ◽

Extracellular Medium ◽

The Stability ◽

Hydrolysis Of ◽

Reduced Activity

The substrate-induced inactivation of beta-lactamase I from Bacillus cereus 569/H has been studied. Both the wild-type enzyme and mutants have been used. The kinetics follow a branched pathway of the type recently analysed [Waley (1991) Biochem. J. 279, 87-94]. The substrate cloxacillin (a penicillin) formed an acyl-enzyme (characterized by m.s.), and it was probably the instability of this intermediate that brought about inactivation. A disulphide bond was introduced into beta-lactamase I (the wild-type enzyme lacks this bond) by site-directed mutagenesis: Ala-77 and Ala-123 were replaced by cysteine. Spontaneous oxidation yielded the disulphide. The activity of this newly cross-linked enzyme was a little diminished, but the stability towards inactivation by cloxacillin was not increased. A second mutant of beta-lactamase I was studied: this mutant lacked the first 17 residues, i.e. the first alpha-helix. The mutant had reduced activity towards ordinary (non-inactivating) substrates and no hydrolysis of cloxacillin could be detected. These mutant enzymes were expressed in Bacillus subtilis, and were purified from the extracellular medium.

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Catalytic mechanism of a family 3 β-glucosidase and mutagenesis study on residue Asp-247

Biochemical Journal ◽

10.1042/bj3550835 ◽

2001 ◽

Vol 355 (3) ◽

pp. 835-840 ◽

Cited By ~ 25

Author(s):

Yaw-Kuen LI ◽

Jiunly CHIR ◽

Fong-Yi CHEN

Keyword(s):

Amino Acid ◽

Essential Amino Acid ◽

Catalytic Mechanism ◽

Bond Cleavage ◽

Isotope Effects ◽

Kinetic Isotope Effects ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Wild Type Enzyme ◽

Kinetic Isotope

A family 3 β-glucosidase (EC 3.2.1.21) from Flavobacterium meningosepticum has been cloned and overexpressed. The mechanistic action of the enzyme was probed by NMR spectroscopy and kinetic investigations, including substrate reactivity, secondary kinetic isotope effects and inhibition studies. The stereochemistry of enzymic hydrolysis was identified as occurring with the retention of an anomeric configuration, indicating a double-displacement reaction. Based on the kcat values with a series of aryl glucosides, a Bronsted plot with a concave-downward shape was constructed. This biphasic behaviour is consistent with a two-step mechanism involving the formation and breakdown of a glucosyl–enzyme intermediate. The large Bronsted constant (β =-0.85) for the leaving-group-dependent portion (pKa of leaving phenols > 7) indicates substantial bond cleavage at the transition state. Secondary deuterium kinetic isotope effects with 2,4-dinitrophenyl β-D-glucopyanoside, o-nitrophenyl β-D-glucopyanoside and p-cyanophenyl β-D-glucopyanoside as substrates were 1.17±0.02, 1.19±0.02 and 1.04±0.02 respectively. These results support an SN1-like mechanism for the deglucosylation step and an SN2-like mechanism for the glucosylation step. Site-directed mutagenesis was also performed to study essential amino acid residues. The activities (kcat/Km) of the D247G and D247N mutants were 30000- and 200000-fold lower respectively than that of the wild-type enzyme, whereas the D247E mutant retained 20% of wild-type activity. These results indicate that Asp-247 is an essential amino acid. It is likely that this residue functions as a nucleophile in the reaction. This conclusion is supported by the kinetics of the irreversible inactivation of the wild-type enzyme by conduritol-B-epoxide, compared with the much slower inhibition of the D247E mutant and the lack of irreversible inhibition of the D247G mutant.

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Identification of Essential Amino Acid Residues of the NorM Na+/Multidrug Antiporter in Vibrio parahaemolyticus

Journal of Bacteriology ◽

10.1128/jb.187.5.1552-1558.2005 ◽

2005 ◽

Vol 187 (5) ◽

pp. 1552-1558 ◽

Cited By ~ 69

Author(s):

Masato Otsuka ◽

Makoto Yasuda ◽

Yuji Morita ◽

Chie Otsuka ◽

Tomofusa Tsuchiya ◽

...

Keyword(s):

Amino Acid ◽

Ethidium Bromide ◽

Vibrio Parahaemolyticus ◽

Drug Transport ◽

Site Directed Mutagenesis ◽

Amino Acid Residues ◽

Acidic Amino Acid ◽

Wild Type ◽

Transmembrane Region ◽

Mutant Proteins

ABSTRACT NorM is a member of the multidrug and toxic compound extrusion (MATE) family and functions as a Na+/multidrug antiporter in Vibrio parahaemolyticus, although the underlying mechanism of the Na+/multidrug antiport is unknown. Acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM are conserved in one of the clusters of the MATE family. In this study, we investigated the role(s) of acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM by site-directed mutagenesis. Wild-type NorM and mutant proteins with amino acid replacements D32E (D32 to E), D32N, D32K, E251D, E251Q, D367A, D367E, D367N, and D367K were expressed and localized in the inner membrane of Escherichia coli KAM32 cells, while the mutant proteins with D32A, E251A, and E251K were not. Compared to cells with wild-type NorM, cells with the mutant NorM protein exhibited reduced resistance to kanamycin, norfloxacin, and ethidium bromide, but the NorM D367E mutant was more resistant to ethidium bromide. The NorM mutant D32E, D32N, D32K, D367A, and D367K cells lost the ability to extrude ethidium ions, which was Na+ dependent, and the ability to move Na+, which was evoked by ethidium bromide. Both E251D and D367N mutants decreased Na+-dependent extrusion of ethidium ions, but ethidium bromide-evoked movement of Na+ was retained. In contrast, D367E caused increased transport of ethidium ions and Na+. These results suggest that Asp32, Glu251, and Asp367 are involved in the Na+-dependent drug transport process.

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