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1.
Identification and characterization of the maltose permease in genetically defined Saccharomyces strain 总被引:3,自引:2,他引:1
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Y S Chang R A Dubin E Perkins C A Michels R B Needleman 《Journal of bacteriology》1989,171(11):6148-6154
Saccharomyces yeasts ferment several alpha-glucosides including maltose, maltotriose, turanose, alpha-methylglucoside, and melezitose. In the utilization of these sugars transport is the rate-limiting step. Several groups of investigators have described the characteristics of the maltose permease (D. E. Kroon and V. V. Koningsberger, Biochim. Biophys. Acta 204:590-609, 1970; R. Serrano, Eur. J. Biochem. 80:97-102, 1977). However, Saccharomyces contains multiple alpha-glucoside transport systems, and these studies have never been performed on a genetically defined strain shown to have only a single permease gene. In this study we isolated maltose-negative mutants in a MAL6 strain and, using a high-resolution mapping technique, we showed that one class of these mutants, the group A mutants, mapped to the MAL61 gene (a member of the MAL6 gene complex). An insertion into the N-terminal-coding region of MAL61 resulted in the constitutive production of MAL61 mRNA and rendered the maltose permease similarly constitutive. Transformation by high-copy-number plasmids containing the MAL61 gene also led to an increase in the maltose permease. A deletion-disruption of MAL61 completely abolished maltose transport activity. Taken together, these results prove that this strain has only a single maltose permease and that this permease is the product of the MAL61 gene. This permease is able to transport maltose and turanose but cannot transport maltotriose, alpha-methylglucoside, or melezitose. The construction of strains with only a single permease will allow us to identify other maltose-inducible transport systems by simple genetic tests and should lead to the identification and characterization of the multiple genes and gene products involved in alpha-glucoside transport in Saccharomyces yeasts. 相似文献
2.
3.
Melibiose permease of Escherichia coli: mutation of aspartic acid 55 in putative helix II abolishes activation of sugar binding by Na+ ions 总被引:1,自引:0,他引:1
T Pourcher M Deckert M Bassilana G Leblanc 《Biochemical and biophysical research communications》1991,178(3):1176-1181
An aspartic residue (Asp55) located in the putative transmembrane alpha-helix II of the melibiose(mel) permease of Escherichia coli was replaced by Cys using oligonucleotide-directed, site-specific mutagenesis. Although D55C permease is expressed at 0.7 times the level of wild type permease, the mutated mel permease loses the ability to catalyse Na+ or H+ coupled melibiose transport against a concentration gradient. (3H) p-nitrophenyl-alpha-D-galactoside (NPG) binding studies demonstrated that D55C permease binds the sugar co-substrate but Na+ (or Li+) ions do no longer enhance the affinity of D55C permease for the co-transported sugar. In addition sugar binding on D55C permease but not on wild type permease is inactivated by sulfhydryl reagents and the inhibition protected by an excess of melibiose. These observations suggest 1) that the negatively-charged Asp55 residue, expected to be within the membrane embedded domain near the NH2 extremity of mel permease, is in or near the Na(+)-binding site and 2) that the cation and sugar binding sites may be overlapping. 相似文献
4.
Binding of p-nitrophenyl alpha-D-galactopyranoside to lac permease of Escherichia coli 总被引:2,自引:0,他引:2
Binding of the substrate analogue p-nitrophenyl alpha-D-galactopyranoside (NPG) to lac permease of Escherichia coli in different membrane preparations was investigated. Binding was assayed with an improved version of the centrifugation technique introduced by Kennedy et al. [Kennedy, E.P., Rumley, M.V., Armstrong, J.B. (1974) J. Biol. Chem. 249, 33-37]. Two binding sites for NPG were found with dissociation constants of about 16 microM and 1.6 mM at pH 7.5 and room temperature. With purified lac permease reconstituted into proteoliposomes, it could be shown that one permease molecule binds two substrate molecules. Oxidation of lac permease with the lipophilic quinone plumbagin or alkylation with the sulfhydryl reagent N-ethylmaleimide caused a 12-fold increase in the first dissociation constant. The second dissociation constant seemed to be increased as well, but its value could not reliably be estimated. Ethoxyformylation of lac permease with diethyl pyrocarbonate totally abolished NPG binding. The implications of these results for the catalytic performance of the enzyme are discussed. 相似文献
5.
Molecular Analysis of Maltotriose Active Transport and Fermentation by Saccharomyces cerevisiae Reveals a Determinant Role for the AGT1 Permease
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Sergio L. Alves Jr. Ricardo A. Herberts Claudia Hollatz Debora Trichez Luiz C. Miletti Pedro S. de Araujo Boris U. Stambuk 《Applied microbiology》2008,74(5):1494-1501
Incomplete and/or sluggish maltotriose fermentation causes both quality and economic problems in the ale-brewing industry. Although it has been proposed previously that the sugar uptake must be responsible for these undesirable phenotypes, there have been conflicting reports on whether all the known α-glucoside transporters in Saccharomyces cerevisiae (MALx1, AGT1, and MPH2 and MPH3 transporters) allow efficient maltotriose utilization by yeast cells. We characterized the kinetics of yeast cell growth, sugar consumption, and ethanol production during maltose or maltotriose utilization by several S. cerevisiae yeast strains (both MAL constitutive and MAL inducible) and by their isogenic counterparts with specific deletions of the AGT1 gene. Our results clearly showed that yeast strains carrying functional permeases encoded by the MAL21, MAL31, and/or MAL41 gene in their plasma membranes were unable to utilize maltotriose. While both high- and low-affinity transport activities were responsible for maltose uptake from the medium, in the case of maltotriose, the only low-affinity (Km, 36 ± 2 mM) transport activity was mediated by the AGT1 permease. In conclusion, the AGT1 transporter is required for efficient maltotriose fermentation by S. cerevisiae yeasts, highlighting the importance of this permease for breeding and/or selection programs aimed at improving sluggish maltotriose fermentations. 相似文献
6.
F.E.M. Cousseau S.L. Alves Jr D. Trichez B.U. Stambuk 《Letters in applied microbiology》2013,56(1):21-29
The genome from the Saccharomyces pastorianus industrial lager brewing strain Weihenstephan 34/70, a natural Saccharomyces cerevisiae/Saccharomyces eubayanus hybrid, indicated the presence of two different maltotriose transporter genes: a new gene in the S. eubayanus subgenome with 81% of homology to the AGT1 permease from S. cerevisiae, and an amplification of the S. eubayanus MTY1 maltotriose permease previously identified in S. pastorianus yeasts. To characterize these S. eubayanus transporter genes, we used a S. cerevisiae strain deleted in the AGT1 permease and introduced the desired permease gene(s) into this locus through homologous recombination. Our results indicate that both the MTY1 and AGT1 genes from the S. eubayanus subgenome encode functional maltotriose transporters that allow fermentation of this sugar by yeast cells, despite their apparent differences in the kinetics of maltotriose‐H+ symport activity. The presence of two maltotriose transporters in the S. eubayanus subgenome not only highlights the importance of sugar transport for efficient maltotriose utilization by industrial yeasts, but these new genes can be used in breeding and/or selection programs aimed at increasing yeast fitness for the efficient fermentation of brewer's wort. 相似文献
7.
Isolation and characterization of thiodigalactoside-resistant mutants of the lactose permease which possess an enhanced recognition for maltose 总被引:5,自引:0,他引:5
In the current study, lactose permease mutants were isolated which exhibited an enhanced recognition for maltose (an alpha-glucoside) but a diminished recognition for thiodigalactoside, TDG (a beta-galactoside). Maltose/TDGR mutants were obtained from four different parental strains encoding either a wild-type permease (pTE18), a mutant lactose permease which recognizes maltose (pB15) or mutant lactose permeases which recognize maltose but are resistant to inhibition by cellobiose (pTG and pBA). A total of 27 independent mutants were isolated: 12 from pTE18, 10 from pB15, 3 from pTG, and 2 from pBA. DNA sequencing of the 27 mutants revealed that the mutants contain single base pair substitutions within the lac Y gene which result in single amino acid substitutions within the lactose permease. All of the mutants obtained from pTE18, pTG, and pBA involved a change of Tyr-236 to histidine, phenylalanine, or asparagine. From pB15, three different types of mutants were obtained: Tyr-236 to histidine, Ile-303 to phenylalanine, or His-322 to asparagine. When assayed for [14C]maltose transport, the maltose/TDGR mutants were seen to transport maltose significantly faster than the wild type. Furthermore, although TDG was shown to inhibit the uptake of maltose in the four parental strains, all of the mutant strains exhibited a dramatic resistance to TDG inhibition. Most of the maltose/TDGR mutants were also shown to be very defective in the transport of lactose. However, certain mutants (i.e., Asn-322) exhibited moderate lactose transport activity. Finally, it was observed that all of the mutant strains were unable to facilitate the uphill accumulation of beta-methylthiogalactopyranoside. The locations of the amino acid substitutions are discussed with regard to their possible role in sugar recognition. 相似文献
8.
An analysis of the side chain requirement at position 177 within the lactose permease which confers the ability to recognize maltose. 总被引:1,自引:0,他引:1
In previous work (Brooker, R. J., and Wilson, T. H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3959-3963), lactose permease mutants were isolated which possessed an enhanced recognition for maltose. In some of these mutants, the wild-type alanine residue at position 177 was changed to valine or threonine. To gain further insight into the side chain requirement at position 177 that confers maltose recognition, further substitutions of isoleucine, leucine, phenylalanine, proline, and serine have been made via site-directed mutagenesis. Permeases containing alanine or serine exhibited poor maltose recognition whereas those containing isoleucine, leucine, phenylalanine, proline, or valine showed moderate or good recognition. As far as galactosides are concerned, the Val-177, Pro-177, and Ser-177 mutants were able to transport lactose as well as, or slightly better than, the wild-type strain. The other mutants displayed moderately reduced levels of lactose transport. For example, the Phe-177 mutant, which was the most defective, showed a level of downhill transport which was approximately 20% that of the wild-type strain. In uphill transport assays, all of the position 177 mutants were markedly defective in their ability to accumulate beta-D-thiomethylgalactopyranoside against a concentration gradient. Finally, the position 177 mutants were analyzed for their ability to catalyze an H+ leak. Interestingly, even though the wild-type permease does not leak H+ across the bacterial membrane, all of the position 177 mutants were shown to transport H+ in the absence of sugars. For most of the mutants, this H+ leak was blocked by the addition of beta-D-thiodigalactoside. Overall, these results are discussed with regard to the effects of position 177 substitutions on the sugar recognition site and H+ transport. 相似文献
9.
The double mutant of the lactose permease containing Val177/Asn319 exhibits proton leakiness by two pathways (see Brooker, R. J. (1991) J. Biol Chem. 266, 4131-4138). One type of H+ leakiness involves the uncoupled influx of H+ (leak A pathway) while a second type involves the coupled influx of H+ and galactosides in conjunction with uncoupled galactoside efflux (leak B pathway). In the current study, 14 independent lactose permease mutants were isolated from the Val177/Asn319 parent which were resistant to thiodigalactoside growth inhibition but retained the ability to transport maltose. All of these mutants contained a third mutation (besides Val177/Asn319) at one of two sites. Eight of the mutants had Ile303 changed to Phe, while six of the mutants had Tyr236 changed to Asn or His. Each type of triple mutant was characterized with regard to sugar transport, H+ leakiness, and sugar specificity. Like the parental strain, all three types of triple mutant showed moderate rates of downhill lactose transport and were defective in the uphill accumulation of sugars. However, with regard to proton leakiness, the triple mutants fell into two distinct categories. The mutant containing Phe303 was generally less H+ leaky than the parent either via the leak A or leak B pathway. In contrast, the triple mutants containing position 236 substitutions (Asn or His) were actually more H+ leaky via the leak A pathway and exhibited similar H+ leakiness via the leak B pathway at high thiodigalactoside concentrations. The ability of the position 236 mutants to grow better than the parent in the presence of low concentrations of thiodigalactoside appears to be due to a decrease in affinity for this particular sugar rather than a generalized defect in H+ leakiness. Finally, the triple mutants showed a sugar specificity profile which was different from either the Val177/Asn319 parent, the single Val177 mutant, or the wild-type strain. These results are discussed with regard to the effects of mutations on both the sugar and H+ transport pathways. 相似文献
10.
AIMS: To enhance the fermentation of maltotriose by industrial Saccharomyces cerevisiae strains. METHODS AND RESULTS: The capability to ferment maltotriose by an industrial yeast strain that uses this sugar aerobically was tested in shake flasks containing rich medium. While the presence of maltose in the medium did not improve maltotriose fermentation, enhanced and constitutive expression of the AGT1 permease not only increased the uptake of maltotriose, but allowed efficient maltotriose fermentation by this strain. Supplementation of the growth medium with 20 mmol magnesium l(-1) also increased maltotriose fermentation. CONCLUSIONS: Over expression of the AGT1 permease and magnesium supplementation improved maltotriose fermentation by an industrial yeast strain that respired but did not ferment this sugar. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes to the elucidation of the roles of the AGT1 permease and nutrients in the fermentation of all sugars present in starch hydrolysates, a highly desirable trait for several industrial yeasts. 相似文献
11.
Temperature-sensitive mutants of Escherichia coli affecting beta-galactoside transport 总被引:3,自引:3,他引:0
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Six different temperature-sensitive (ts) mutants have been isolated which have parental beta-galactoside permease levels at low temperatures but have decreased permease levels when grown at high temperatures. These mutants were derived from Escherichia coli ML308 (lacI(-)Y(+)Z(+)A(+)). After N-methyl-N'-nitro-N'-nitro-soguanidine mutagenesis, ampicillin was used to select for cells unable to grow on low lactose concentrations at 42 C. Temperature-sensitive mutants were assayed for galactoside permease activity after growth in casein hydrolysate medium at 25 or 42 C by measuring both radioactive methylthio-beta-d-galactoside uptake and in vivo o-nitrophenyl-beta-d-galactoside hydrolysis. The six conditional isolates have decreased levels of galactoside permease which are correlated with decreased growth rates at elevated temperatures. The low permease levels are not due to a temperature labile lacY gene product but rather to a temperature labile synthesis rate of functional permease. Some of the mutants exhibit a ts increase in permeability as shown by the increased leakage of intracellular beta-galactosidase and by the increased rate of in vivo o-nitrophenyl-beta-d-galactoside hydrolysis via the nonpermease mediated entry mechanism. Preliminary evidence indicates that transport in general is decreased in these mutants, yet there is some specificity in the mutational lesion since glucoside transport is unaffected. All these observations suggest that these mutants have ts alterations in membrane synthesis which results in pleiotropic effects on various membrane functions. 相似文献
12.
R J Brooker 《The Journal of biological chemistry》1991,266(7):4131-4138
The sugar specificity mutants of the lactose permease containing Val177 or Val177/Asn319 were analyzed with regard to their ability to couple H+ and sugar co-transport. Both mutants were able to transport lactose downhill to a significant degree. The Val177 mutant was partially defective in the active accumulation of galactosides, whereas the Val177/Asn319 mutant was completely defective in the uphill accumulation of sugars. With regard to coupling, the Val177 mutant was shown to catalyze the uncoupled transport of H+ to a substantial degree. This led to a decrease in the H+ electrochemical gradient under aerobic conditions and also resulted in faster H+ uptake when a transient H+ electrochemical gradient was generated under anaerobic conditions. Interestingly, galactosides were shown to diminish the rate of uncoupled H+ transport in the Val177 strain. The Val177/Asn319 strain also catalyzed uncoupled H+ transport, but to a lesser degree than the single Val177 mutant. In addition, the Val177/Asn319 mutant was shown to transport galactosides with or without H+. The observed H+/lactose stoichiometry was 0.30 in the double mutant compared to 0.98 in the wild-type strain. When an H+ electrochemical gradient was generated across the membrane, the Val177/Asn319 mutant permease was shown to facilitate an extremely rapid net H+ leak if nonmetabolizable galactosides had been equilibrated across the membrane. The mechanism of this leak is consistent with a circular pathway involving H+/galactoside influx and uncoupled galactoside efflux. The magnitude of the H+ leak in the presence of nonmetabolizable galactosides was so great in the double mutant that low concentrations of certain galactosides (i.e. 0.5 mM thiodigalactoside) resulted in a complete inhibition of growth. These results are discussed with regard to the possibility that cation and sugar binding to the lactose permease may involve a direct physical coupling at a common recognition site. 相似文献
13.
Within the lactose permease, an arginine residue is found on a transmembrane segment at position 302. Based upon the effects of mutations at or in the vicinity of Arg-302, this residue has been implicated to be involved with H+ and/or sugar recognition. To further elucidate the role of this residue, we have substituted Arg-302 with serine, histidine, and leucine via site-directed mutagenesis. All three of these substitutions result in an impaired ability to transport galactosides as evidenced by their poor growth on minimal plates supplemented with lactose or melibiose. Furthermore, in vitro transport assays revealed substantial alterations in the kinetic constants for downhill lactose transport. The wild-type strain exhibited a Km for lactose transport of 0.30 mM and a Vmax of 267 nmol of lactose/min.mg of protein. The Ser-302, His-302, and Leu-302 were observed to have Km values of 0.18, 2.3, and 2.8 mM, and Vmax values of 11.6, 56.4, and 22.0 nmol of lactose/min.mg of protein, respectively. In uphill transport assays, all three mutants were unable to accumulate beta-methyl-D-thiogalactoside. However, both the Ser-302 and His-302 mutants were able to accumulate lactose against a concentration gradient. During H+ transport assays, all three mutants were shown to transport H+ in conjunction with thiodigalactoside. In addition, the Ser-302 and His-302 strains exhibited small alkalinizations upon the addition of lactose. However, for the Leu-302 mutant, the addition of lactose did not result in a significant level of H+ transport. Finally, experiments were conducted which were aimed at measuring the ability of the mutant permeases to catalyze an H+ leak. In this regard, a comparison was made between the wild-type and mutant strains concerning their steady state pH gradient and their rates of H+ influx following oxygen pulses. The results of these experiments suggest that mutations at position 302 cause a sugar-dependent H+ leak. 相似文献
14.
Five lacY mutants with amber stop codons at known positions were each placed into 12 different suppressor strains. The 60 amino acid substitutions obtained in this manner were tested for growth on lactose-minimal medium plates and for transport of lactose, melibiose, and thiomethylgalactoside. Most of the amino acid substitutions in the regions of the putative loops (between transmembrane alpha helices) resulted in a reasonable growth rate on lactose with moderate-to-good transport activity. In one strain (glycine substituted for Trp-10), abnormal sugar recognition was found. The substitution of proline for Trp-33 (in the region of the first alpha helix) showed no activity, while four additional substitutions (lysine, leucine, cysteine, and glutamic acid) showed low activity. Altered sugar specificity was observed when Trp-33 was replaced by serine, glutamine, tyrosine, alanine, histidine, or phenylalanine. It is concluded that Trp-33 may be involved directly or indirectly in sugar recognition. 相似文献
15.
Enzymes II of the phosphotransferase system do not catalyze sugar transport in the absence of phosphorylation. 总被引:13,自引:9,他引:4
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In Salmonella typhimurium, glucose, mannose, and fructose are normally transported and phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system. We have investigated the transport of these sugars and their non-metabolizable analogs in mutant strains lacking the phospho-carrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system, the enzymes I and HPr, to determine whether the sugar-specific, membrane-bound components of the phosphonenolpyruvate: sugar phosphotransferase system, the enzymes II, can catalyze the uptake of these sugars in the absence of phosphorylation. This process does not occur. We have also isolated mutant strains which lack enzyme I and HPr, but have regained the ability to grow on mannose or fructose. These mutants contained elevated levels of mannokinase (fructokinase). In addition, growth on mannose required constitutive synthesis of the galactose permease. When strains were constructed which lacked the galactose permease, they were unable to grow even on high concentrations of mannose, although elevated levels of mannokinase (fructokinase) were present. These results substantiate the conclusion that the enzymes II of the phosphoenolpyruvate:sugar phosphotransferase system are unable to carry out facilitated diffusion. 相似文献
16.
Uptake of cell wall peptides by Salmonella typhimurium and Escherichia coli. 总被引:21,自引:9,他引:12
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During bacterial growth, cell wall peptides are released from the murein and reused for the synthesis of new cell wall material. Mutants defective in peptide transport were unable to reutilize cell wall peptides, demonstrating that these peptides are taken up intact into the cytoplasm prior to reincorporation into murein. Furthermore, cell wall peptide recycling was shown to play an important physiological role; peptide transport mutants which were unable to recycle these peptides showed growth defects under appropriate conditions. Using mutants specifically defective in each of the three peptide transport systems, we showed that the uptake of cell wall peptides was mediated solely by the oligopeptide permease (Opp) and that neither the dipeptide permease (Dpp) nor the tripeptide permease (Tpp) played a significant role in this process. Our data indicate that the periplasmic oligopeptide-binding protein has more than one substrate-binding site, each with different though overlapping specificities. 相似文献
17.
Zastrow CR Hollatz C de Araujo PS Stambuk BU 《Journal of industrial microbiology & biotechnology》2001,27(1):34-38
Maltotriose, the second most abundant sugar of brewer's wort, is not fermented but is respired by several industrial yeast
strains. We have isolated a strain capable of growing on a medium containing maltotriose and the respiratory inhibitor, antimycin
A. This strain produced equivalent amounts of ethanol from 20 g l−1 glucose, maltose, or maltotriose. We performed a detailed analysis of the rates of active transport and intracellular hydrolysis
of maltotriose by this strain, and by a strain that does not ferment this sugar. The kinetics of sugar hydrolysis by both
strains was similar, and our results also indicated that yeast cells do not synthesize a maltotriose-specific α-glucosidase.
However, when considering active sugar transport, a different pattern was observed. The maltotriose-fermenting strain showed
the same rate of active maltose or maltotriose transport, while the strain that could not ferment maltotriose showed a lower
rate of maltotriose transport when compared with the rates of active maltose transport. Thus, our results revealed that transport
across the plasma membrane, and not intracellular hydrolysis, is the rate-limiting step for the fermentation of maltotriose
by these Saccharomyces cerevisiae cells. Journal of Industrial Microbiology & Biotechnology (2001) 27, 34–38.
Received 13 January 2001/ Accepted in revised form 29 May 2001 相似文献
18.
Role of cyclic-AMP-dependent protein kinase in catabolite inactivation of the glucose and galactose transporters in Saccharomyces cerevisiae. 总被引:7,自引:2,他引:5
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The derepressed high-affinity glucose transport system and the induced galactose transport system are catabolite inactivated when cells with these transport systems are incubated with glucose. The role of the cyclic AMP cascade in the catabolite inactivation of these transport systems was shown by using mutants affected in the activity of cyclic-AMP-dependent protein kinase (cAPK). In tpk1(w) mutants with reduced cAPK activity, the sugar transport systems were expressed but were not catabolite inactivated. In bcy1 mutants with unbridled cAPK activity resulting from a defective regulatory subunit, the transport systems were absent or present at low levels. 相似文献
19.
20.
Cysteine scanning mutagenesis of the N-terminal 32 amino acid residues in the lactose permease of Escherichia coli. 总被引:4,自引:3,他引:1
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M. Sahin-Tth B. Persson J. Schwieger P. Cohan H. R. Kaback 《Protein science : a publication of the Protein Society》1994,3(2):240-247
Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in the hydrophilic N-terminus and the first putative transmembrane helix was systematically replaced with Cys (from Tyr-2 to Trp-33). Twenty-three of 32 mutants exhibit high lactose accumulation (70-100% or more of C-less), and an additional 8 mutants accumulate to lower but highly significant levels. Surprisingly, Cys replacement for Gly-24 or Tyr-26 yields fully active permease molecules, and permease with Cys in place of Pro-28 also exhibits significant transport activity, although previous mutagenesis studies on these residues suggested that they may be required for lactose transport. As expected, Cys replacement for Pro-31 completely inactivates, in agreement with previous findings indicating that "helix-breaking" propensity at this position is necessary for full activity (Consler TG, Tsolas O, Kaback HR, 1991, Biochemistry 30:1291-1297). Twenty-nine mutants are present in the membrane in amounts comparable to C-less permease, whereas membrane levels of mutants Tyr-3-->Cys and Phe-12-->Cys are slightly reduced, as judged by immunological techniques. Dramatically, mutant Phe-9-->Cys is hardly detectable when expressed from the lac promoter/operator at a relatively low rate, but is present in the membrane in a stable form when expressed at a high rate from the T7 promoter. Finally, studies with N-ethylmalemide show that 6 Cys-replacement mutants that cluster at the C-terminal end of putative helix I are inactivated significantly.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献