A valine-resistant mutant ofArabidopsis thaliana displays an acetolactate synthase with altered feedback control

A valine-resistant mutant line, VAL-2, ofArabidopsis thaliana (L.) Heynh. was identified by screening M 2 populations of ethylmethane-sulfonate-mutagenized seeds. The resistance was found to be due to a single, dominant, nuclear gene mutation. Assay of acetolactate synthase (ALS) indicated that the valine resistance in this mutant is caused by decreased sensitivity of ALS to the branched-chain amino acids, valine, leucine andisoleucine. A two fold decrease in apparentK _m value for pyruvate of the mutant ALS enzyme was detected compared with that of the wild type. The sensitivity of the ALS enzyme to sulfonylurea, imidazolinone and triazolopyrimidine herbicides was not altered in the mutant. At the plant growth level the mutant was also resistant to valine plus leucine, but was sensitive to leucine orisoleucine alone. The mutant gene,var1, maps, or is very closely linked, toCSR1, the gene encoding acetolactate synthase inArabidopsis.Abbreviations ALS acetolactate synthase - BCAA branched-chain amino acid - CS chlorsulfuron - IM imidazolinone - SU sulfonylurea - TP triazolopyrimidine We thank Dr. George W. Haughn for providing Arabidopsis lines MSU12, MSU15, MSU21, MSU22 and MSU23. This work was supported by a Research Grant from the Natural Sciences and Engineering Research Council of Canada to J.K., K.W. is grateful for a University of Saskatchewan Graduate Scholarship.     

Conformational effects of amino acid substitutions in the P-glycoprotein of themdr 1 gene

The P-glycoprotein of themdr 1 gene is responsible for the phenomenon of multidrug resistance in human cells. The presumed drug-binding site of the wild-type P-glycoprotein contains a glycine at position 185. A mutant P-glycoprotein which contains valine at this position causes cells to retain resistance to colchichine, but to lose cross-resistance to other drugs such as the chemotherapeutic agents vinblastine and Adriamycin. This has been hypothesized to be due to a conformational change in the protein induced by the amino acid substitution. Using conformational energy analysis, we have determined the allowed three-dimensional structures for the wild-type and mutant proteins in the region of position 185. The results indicate that the wild-type protein adopts a unique left-handed conformation at position 185 which is energetically unfavorable for the protein withl-amino acids (including valine) at this position. This conformational change induced by amino acid substitutions for Gly 185 could explain the differences in binding to the P-glycoprotein of various drugs and, hence, the differences in drug resistance exhibited by various cell lines expressing these proteins.     

Isolation and characterization of plant genes coding for acetolactate synthase, the target enzyme for two classes of herbicides

Acetolactate synthase (ALS) is the first common enzyme in the biosynthetic pathways to valine, isoleucine, and leucine. It is the target of two structurally unrelated classes of herbicides, the sulfonylureas and the imidazolinones. Genomic clones encoding ALS have been isolated from the higher plants Arabidopsis thaliana and Nicotiana tabacum, using a yeast ALS gene as a heterologous hybridization probe. Clones were positively identified by the homology of their deduced amino acid sequences with those of yeast and bacterial ALS isozymes. The tobacco and Arabidopsis ALS genes have approximately 70% nucleotide homology, and encode mature proteins which are approximately 85% homologous. Little homology is seen between the amino acid sequences of the presumptive N-terminal chloroplast transit peptides. Both plant genes lack introns. The tobacco ALS gene was isolated from a line of tobacco which is resistant to the sulfonylurea herbicides due to an alteration in ALS. The tobacco gene which was isolated codes for an ALS that is sensitive to the herbicides, as assayed by transformation of the gene into sensitive tobacco cells.     

Sulfonylurea-resistant mutants and natural tolerance of cyanobacteria

The herbicide sulfometuron methyl (SM) inhibited the growth of the cyanobacterium Synechococcus sp. PCC7942, but not of Synechocystis sp. PCC6714. The inhibitory effect was alleviated by the simultaneous addition of valine, leucine and isoleucine. SM resistant mutants were isolated from Synechococcus 7942, two types of which were further analysed. In these mutants, SM3/20 and SM2/32, the activity of acetolactate synthase (ALS) — a key enzyme in the biosynthesis of branched-chain amino acids —appeared 2600- and 300-fold, respectively, more resistant to SM than that of their wild type. Strain SM2/32 also exhibited a low level of ALS activity. Although the growth of the latter mutant was extremely inhibited by valine, the sensitivity of its ALS activity to feed-back inhibition by the amino acid was unaltered. At high concentrations valine inhibited growth of the wild type strains and of the mutant SM3/20. Isoleucine alleviated the valine-induced growth inhibition. Unlike that of Synechococcus 7942, the ALS activity of Synechocystis was found to tolerate high concentrations (100-fold) of the herbicide. The study confirms that the SM mutations are correlated with a cyanobacterial ilv gene.Abbreviations ALS acetolactate synthase; ile, isoleucine - leu leucine - NTG N-methyl-N-nitro-N-nitrosoguanidine - SM sulfometuron methyl - SM^r sulfometuron methyl resistant - val valine     

Leucine and serine induce mecillinam resistance in Escherichia coli.

Summary We have previously shown that resistance to the -lactam mecillinam in Escherichia coli can be brought about by a high ppGpp pool, as observed under conditions of partial amino acid starvation and ReIA-dependent induction of the stringent response. We show here that our E. coli wild-type strain, which is sensitive to mecillinam on minimal glucose plates, becomes resistant in the presence of lleucine or L-serine (or cysteine, which inactivates the antibiotic). The resistance, which is not a transient effect and does not depend on the physiological state of the cells when plated, is specific for mecillinam and is reversed by the presence of isoleucine and valine in the medium. At least in the case of serine, the resistance is ReIA-dependent. We conclude that the presence of leucine and serine in the growth medium cause partial starvation for isoleucine/valine, leading to induction of the stringent response and concomitant resistance to mecillinam.     

Molecular analysis of the acetolactate synthase gene of Chlamydomonas reinhardtii and development of a genetically engineered gene as a dominant selectable marker for genetic transformation

Genomic and cDNA clones of the acetolactate synthase (ALS) gene of Chlamydomonas reinhardtii have been isolated from a mutant, c85-20 (Hartnett et al., 1987), that is resistant to high concentrations of sulfometuron methyl (SMM) and related sulfonylurea herbicides. Comparison of the ALS gene sequences from the wild-type and the SMM resistant (SMMr) strains revealed two amino acid differences in the mature enzyme, a lysine to threonine change at position 257 (K257T) and a leucine to valine change at position 294 (L294V). Transformation of wild-type C. reinhardtii with the mutant ALS gene produced no transformants with ability to grow in the presence of a minimum toxic concentration of SMM (3 microm). Substitution of the ALS promoter with the promoter of the C. reinhardtii Rubisco small subunit gene (RbcS2) permitted recovery of SMMr colonies. In vitro mutagenesis of the wild-type ALS gene to produce various combinations of mutations (K257T, L294V and W580L) indicated that the K257T mutation was necessary and sufficient to confer the SMMr phenotype. Optimum transformation rates were obtained with two constructs (pJK7 and pRP-ALS) in which all introns in the coding region were present. Rates of transformation with construct pJK7 were approximately 2.5 x 10-4 transformants/cell (i.e. one transformant for each of 4000 initial cells) using electroporation and 8.5 x 10-6 transformants/cell using the glass bead vortexing method. These results suggest that pJK7 and pRP-ALS can serve as important additional dominant selectable markers for the genetic transformation of C. reinhardtii.     

Genetic analysis of two new mutations resulting in herbicide resistance in the cyanobacterium Synechcoccus sp. PCC 7002

Two herbicide-resistant strains of the cyanobacterium Synechococcus sp. PCC 7002 are compared to the wild-type with respect to the DNA changes which result in herbicide resstance. The mutations have previously been mapped to a region of the cyanobacterial genome which encodes oneof three copies of psbA, the gene which encodes the 32 kDa Q_b-binding protein also known as D1 (Buzby et al. 1987). The DNA sequence of the wild-type gene was first determined and used as a comparison to that of the mutant alleles. A point mutation at codon 211 in the psbA1 coding locus (TTC) to TCC) results in an amino acid change from phenylalanine to serine in the D1 protein. This mutation confers resistance to atrazine and diuron at seven times and at two times the minimal inhibitory concentration (MIC) for the wild-type, respectively. A mutation at codon 211 resulting in herbicide resistance has not previously been described in the literature. A second point mutation at codon 219 in the psbA1 coding locus (GTA to ATA) results in an amino acid change from valine to isoleucine in the D1 protein. This mutation confers resistance to diuron and atrazine at ten times and at two times the MIC for the wild-type, respectively. An identical codon change conferring similar herbicide resistance patterns has previously been described in Chlamydomonas reinhardtii. The atrazine-resistance phenotype in Synechococcus sp. PCC 7002 was shown to be dominant by plasmid segregation analysis.Abbreviations At ^r atrazine resistance - Du ^r diuron resistance - Km ^r kanamycin resistance - Ap ^r ampicillin resistance - MIC Minimum inhibitory concentration     

Conformational effects of amino acid substitutions in the P-glycoprotein of themdr 1 gene

The P-glycoprotein of themdr 1 gene is responsible for the phenomenon of multidrug resistance in human cells. The presumed drug-binding site of the wild-type P-glycoprotein contains a glycine at position 185. A mutant P-glycoprotein which contains valine at this position causes cells to retain resistance to colchichine, but to lose cross-resistance to other drugs such as the chemotherapeutic agents vinblastine and Adriamycin. This has been hypothesized to be due to a conformational change in the protein induced by the amino acid substitution. Using conformational energy analysis, we have determined the allowed three-dimensional structures for the wild-type and mutant proteins in the region of position 185. The results indicate that the wild-type protein adopts a unique left-handed conformation at position 185 which is energetically unfavorable for the protein withl-amino acids (including valine) at this position. This conformational change induced by amino acid substitutions for Gly 185 could explain the differences in binding to the P-glycoprotein of various drugs and, hence, the differences in drug resistance exhibited by various cell lines expressing these proteins.     

Valine 77 of heterocystous ferredoxin FdxH2 in Anabaena variabilis strain ATCC 29413 is critical for its oxygen sensitivity

Ferredoxins are small iron sulfur proteins necessary for electron donation. FdxH1 and FdxH2 are associated with two different nif gene clusters where they transfer electrons for the reduction of nitrogenase complex. FdxH1 was observed to be stable towards oxygen, whereas, FdxH2 was relatively unstable. We had identified the amino acid involved in oxygen sensitivity of ferredoxin protein using protein modeling. The exchange of valine to leucine at position 77 was critical for ferredoxin proteins in relation to its oxygen sensitivity. This exchange leads to a longer side chain, which inhibits the accessibility of oxygen to the iron sulfur cluster. Site directed mutagenesis and in vitro experiments confirms that valine indeed is involved in the oxygen sensitivity. The exchange of leucine to valine in FdxH1 makes it oxygen unstable. Thus, from the above results we can conclude that the position of leucine at position 77 is critical for oxygen sensitivity of ferredoxin and protein modeling can be used to identify specific amino acids in other oxygen-sensitive proteins.     

Molecular dynamics study of structure and stability of a model coiled coil

This paper employs methods used earlier to study helix propensity in a model α-helix. The methods are extended to simulations of a motif structure of the α-helical coiled coil, i.e., a structure with a simple amino acid sequence, containing only alanine, leucine, and valine, with leucine and valine forming hydrophobic contacts in the helix interface (positions “d” and “a”). Dynamic simulations of the model coiled-coil structure reproduce characteristic features of the coiled-coil motif seen in experimental studies. Free energy simulations were used to assess the change in stability of the model when a leucine pair or a valine pair in the helix interface was replaced with an alanine pair. A leucine pair at position d was found to contribute 3.4 kcal/mol to the stability of the coiled coil relative to an alanine pair, and a valine pair at postion a was found to contribute 0.8 kcal/mol relative to an alanine pair. The value for the leucine pair agrees with reports in two experimental studies with molecules having different amino sequence. The value for the valine pair is reasonable given the smaller size of the valine side chain and the intrinsic low helix propensity of valine. No experimental value was available for comparison. © 1993 Wiley-Liss, Inc.     

Molecular Basis of Imidazolinone Herbicide Resistance in Arabidopsis thaliana var Columbia

Acetolactate synthase (ALS), the first enzyme in the biosynthetic pathway of leucine, isoleucine, and valine, is inhibited by imidazolinone herbicides. To understand the molecular basis of imidazolinone resistance, we isolated the ALS gene from an imazapyr-resistant mutant GH90 of Arabidopsis thaliana. DNA sequence analysis of the mutant ALS gene demonstrated a single-point mutation from G to A at nucleotide 1958 of the ALS-coding sequence. This would result in Ser to Asn substitution at residue 653 near the carboxyl terminal of the matured ALS. The mutant ALS gene was introduced into tobacco using Agrobacterium-mediated transformation. Imidazolinone-resistant growth of transformed calli and leaves of transgenic plants was 100-fold greater than that of nontransformed control plants. The relative levels of imidazolinone-resistant ALS activity correlated with the amount of herbicide-resistant growth in the leaves of transgenic plants. Southern hybridization analysis confirmed the existence of transferred ALS gene in the transformant showing high imazapyr resistance. The results demonstrate that the mutant ALS gene confers resistance to imidazolinone herbicides. This is the first report, to our knowledge, of the molecular basis of imidazolinone resistance in plants.     

Effects of branched-chain amino acids on plasma amino acids in amyotrophic lateral sclerosis

Summary Although the cause of amyotrophic lateral sclerosis (ALS) remains unknown, biological findings suggest that the excitatory amino acid glutamate contributes to the pathogenesis of ALS. In previous studies of ALS, the therapeutic effect of the branched-chain amino acids (BCAAs) leucine, valine and isoleucine has been evaluated. The present study aimed at investigating the acute effect of BCAAs on plasma glutamate levels in ALS patients. Following two oral doses of BCAAs, significantly increased plasma levels were seen for valine (500%), isoleucine (1,377%) and leucine (927%), however the plasma level of glutamate was not affected. The plasma level of several other amino acids (tryptophan, tyrosine, phenylalanine and methionine) were found decreased after oral BCAAs, which may indicate a diminution in the rate of degradation of muscle protein and/or an increase in tissue disposal of amino acids.     

Deregulated branched-chain amino acid synthesis in a Nicotiana plumbaginifolia cell line resistant to valine

A Nicotiana plumbaginifolia cell line able to grow in the presence of high doses of valine was isolated following -rays mutagenesis. The selected clone, named D5R5, showed a growth rate higher than that of wild-type. It was less sensitive also to an equimolar mixture of the three branched-chain amino acids, but did not display cross-resistance to isoleucine and leucine. The increased tolerance was due to neither a reduced valine uptake, nor a modification in the level or sensitivity to feed-back inhibition by valine of the first common enzyme (and the main regulative site) in isoleucine, leucine and valine synthesis, acetohydroxyacid synthase (AHAS). When wild-type cells were fed with valine or equimolar mixtures of the three aminoacids, a decrease in AHAS level was found. On the contrary, the level of extractable AHAS activity from D5R5 cells was significantly less affected by similar treatments, suggesting that some alteration in enzyme modulation mechanism(s) could account for valine resistance.Abbreviations AHAS acetohydroxyacid synthase - BCAA branched-chain amino acid - FAD flavin adenine dinucleotide - ILV equimolar mixture of isoleucine, leucine and valine - TPP thiamine pyrophosphate     

A dominant interfering mutation in RAS1 of Saccharomyces cerevisiae

A mutant allele of RAS1 that dominantly interferes with the wild-type Ras function in the yeast Saccharomyces cerevisiae was discovered during screening of mutants that suppress an ira2 disruption mutation. A single amino acid substitution, serine for glycine at position 22, was found to cause the mutant phenotype. The inhibitory effect of the RAS1 ^Ser22 gene could be overcome either by overexpression of CDC25 or by the ira2 disruption mutation. These results suggest that the RAS1^Ser22 gene product interferes with the normal interaction of Ras with Cdc25 by forming a dead-end complex between Ras1^Ser22 and Cdc25 proteins.     

Amino acid requirements for growth of the rabbit blastocyst in vitro

Ten amino acids, namely, arginine, histidine, lysine, tryptophane, methionine, phenylalanine, leucine, valine, threonine and serine were indispensable for growth of rabbit blastocysts in vitro; others were nonessential. Of all the essential amino acids, arginine and lysine were required in relatively high concentrations, 10^?2 M and 10^?3 M, respectively, for optimum growth. Complete omission of the non-essential amino acids from the medium markedly reduced blastocyst growth. Interaction between serine and glycine demonstrated a partial sparing action on serine by glycine, similar to that observed between methionine and cysteine. The amino acid composition of a culture medium capable of providing continuous and consistent growth of rabbit blastocysts in vitro is described.     

Acetolactate synthases MoIlv2 and MoIlv6 are required for infection‐related morphogenesis in Magnaporthe oryzae

Amino acids are important components in the metabolism of a variety of pathogens, plants and animals. Acetolactate synthase (ALS) catalyses the first common step in leucine, isoleucine and valine biosynthesis, and is the target of several classes of inhibitors. Here, MoIlv2, an orthologue of the Saccharomyces cerevisiae ALS catalytic subunit Ilv2, and MoIlv6, an orthologue of the S. cerevisiae ALS regulatory subunit Ilv6, were identified. To characterize MoILV2 and MoILV6 functions, we generated the deletion mutants ΔMoilv2 and ΔMoilv6. Phenotypic analysis showed that both mutants were auxotrophic for leucine, isoleucine and valine, and were defective in conidial morphogenesis, appressorial penetration and pathogenicity. Further studies suggested that MoIlv2 and MoIlv6 play a critical role in maintaining the balance of intracellular amino acid levels. MoIlv2 and MoIlv6 are both localized to the mitochondria and the signal peptide of MoIlv6 is critical for its localization. In summary, our evidence indicates that MoIlv2 plays a crucial role in isoleucine and valine biosynthesis, whereas MoIlv6 contributes to isoleucine and leucine biosynthesis; both genes are required for fungal pathogenicity. This study indicates the potential of targeting branched‐chain amino acid biosynthesis for anti‐rice blast management.     

The molecular basis of sulfonylurea herbicide resistance in tobacco

The enzyme acetolactate synthase (ALS) is the target enzyme for the sulfonylurea and imidazolinone herbicides. We describe the isolation and characterization of the ALS genes from two herbicide-resistant mutants, C3 and S4-Hra, of Nicotiana tabacum. There are two distinct ALS genes in tobacco which are 0.7% divergent at the amino acid sequence level. The C3 mutant has a single Pro-Gln replacement at amino acid 196 in one ALS gene. This gene is termed the class I gene and is equivalent to the SuRA locus. The S4-Hra mutant has two amino acid changes in the other ALS gene. This gene is termed the class II gene or the SuRB locus. The S4-Hra mutant includes a Pro-Ala substitution at amino acid 196 and a Trp-Leu substitution at amino acid 573. Gene reintroduction experiments have confirmed that these amino acid substitutions are responsible for the herbicide resistance phenotypes. Transgenic plants carrying these genes are highly resistant to sulfonylurea herbicide applications.     

Amino acid fermentation and hydrogen transfer in mixed cultures

Abstract The degradation of the following amino acids was investigated in mixed cultures obtained from a waste water purification plant: aspartate, glutamate, serine, alanine, valine and leucine. Inhibition of sulfate-reducing bacteria in these mixed cultures by molybdate was found to inhibit amino acid degradation. The degradation of serine, alanine, valine and leucine was accelerated considerably by active sulfate reduction. The fermentation of aspartate and glutamate was not stimulated by the presence of sulfate-reducing bacteria. The existence of species which are able to ferment valine and leucine by coupling their oxidation to the reduction of exogenous acetate to butyrate was demonstrated.     

Identification of Des-methyl-DIF-1 Methyltransferase in Dictyostelium purpureum

For the production of D-amino acids using stable N-carbamyl-D-amino acid amidohydrolase (DCase) in an immobilized form, the DCase gene of Agrobacterium sp. KNK712 was mutagenized to increase its enzymatic thermostability. In a search for thermostability-related amino acid sites besides the two known sites of DCase, i.e., the 57th and 203rd amino acids, the new mutant enzyme found, in which the 236th amino acid, valine, had been changed to alanine, showed a 10°C increase in thermostability. These known three thermostability-related amino acids were changed to other amino acids by the PCR technique, and it was proved that the thermostability of the DCase increased when the 57th amino acid of DCase, histidine, was changed to leucine, the 203rd amino acid, proline, to asparagine, glutamate, alanine, isoleucine, histidine, or threonine, and the 236th amino acid, valine, to threonine or serine, in addition to the known mutations.