Mark Levinthal

The acetohydroxy acid synthase III isoenzyme of Escherichia coli K-12: regulation of synthesis by leucine.

Abstract Acetohydroxy acid synthase III (AHAS III) is one of the three isoenzymes which catalyze the condensation reaction for the biosynthesis of the branched chain amino acids in Escherichia coli K-12. The synthesis of this enzyme is repressed by leucine. As a consequence of this regulatory feature, strain PS1035, in which AHAS III is the only AHAS isoenzyme expressed, does not grow in minimal medium containing leucine. The other two branched chain amino acids, isoleucine and valine, do not have regulatory effects on AHAS III synthesis.

Growth inhibition of Escherichia coli K-12 by L-valine: a consequence of a regulatory pattern.

SummaryWe studied the production of the ilvG gene product, the valine resistant acetolactate synthase isoenzyme II, in an ilvO+G+ilvB ilvHI derivative of Escherichia coli K-12. This strain contains mutations in the structural genes for the valine sensitive acetolactate synthase isoenzymes I and III. We find that the ilvG gene is not expressed in this strain when grown with either isoleucine and valine or with isoleucine, leucine and valine, or when limited for either isoleucine or valine. Since we previously found that the ilvG gene is expressed in an ilvO603 containing strain (Favre et al., 1976), we presume that the mechanism by which E. coli K-12 regulates the ilv gene cluster is responsible for the lack of ilvG expression in the ilvO+ strain. The valine sensitivity of E. coli K-12 is a consequence of this regulatory pattern.

A comparative study of the acetohydroxy acid synthase isoenzymes of Escherichia coli K-12

Abstract We studied the properties of the two acetohydroxy acid synthase isoenzymes expressed in wild type Escherichia coli K-12 in two isogenic strains, PS1035 (containing only acetohydroxy acid synthase III) and PS1036 (containing only acetohydroxy acid synthase I). The pH dependence is different for the two enzymes: acetohydroxy acid synthase I shows optimum activity at neutral pH, while acetohydroxy acid synthase III is most active at alkaline pH. Both activities require Mg2+ and thiamine pyrophosphate, but acetohydroxy acid synthase I, as compared to acetohydroxy acid synthase III, has a specific requirement for flavin adenine dinucleotide. Acetohydroxy acid synthase I is also more resistant to valine inhibition but more sensitive to inactivating conditions such as dialysis and temperature. The catalytic role of acetohydroxy acid synthase I in the synthesis of α-acetolactate is characterized by a higher affinity for pyruvate and a lower sensitivity to inhibition by α-ketobutyrate.

The H-NS protein modulates the activation of the ilvIH operon of Escherichia coli K12 by Lrp, the leucine regulatory protein.

The H-NS protein, the product of the hns gene, plays a central role in the cellular response of bacteria to environmental stresses such as modification of osmolarity and temperature. The leucine regulatory protein (Lrp) controls a wide array of operons both as an activator (e.g. ilvIH) and as a repressor. We demonstrate that H-NS can decrease the activity of Lrp in stationary phase and under conditions of high osmolarity. Strains containing hns mutations have higher levels of Lrp-activated ilvIH transcription, while strains carrying the hns+ allele on a pBR322 plasmid have lower activity of Lrp-directed ilvIH gene expression.

The role of H-NS in one carbon metabolism

The H-NS protein of Escherichia coli regulates the expression of genes involved in many general processes such as osmoregulation and virulence. More recently, H-NS was shown to exert an effect on ilvIH gene expression in conjunction with the leucine responsive regulatory protein (Lrp). We show that H-NS is involved in the transcriptional regulation of the kbl/tdh operon, which is also Lrp regulated. Insertional inactivation of the hns gene results in two-fold derepression of the kbl/tdh operon. This level of expression is sufficient to suppress the auxotrophic requirements imposed by a glyA mutation. We show that expression of the kbl/tdh operon is temperature controlled and that this control is not mediated through H-NS action as has been shown for some other temperature controlled genes.

Synthesis of the isoleucyl- and valyl-tRNA synthetases and the isoleucine-valine biosynthetic enzymes in a threonine deaminase regulatory mutant of Escherichia coli K-12☆

A mutation in the structural gene for threonine deaminase, ilvA538 , results in lower than normal levels of the isoleucyl, valyl- and leucyl-tRNA synthetases. Moreover, this regulatory mutation decreases the level of expression of the ilv biosynthetic operons and renders their expression non-responsive to limitations of the branched-chain amino acids. In this paper, we present in vitro evidence for the inhibition of isoleucyl- and valyl-tRNA synthetase activity by threonine deaminase and 2-ketobutyrate, the product of the threonine deaminase reaction, through the formation of a high molecular weight complex of the three molecules. Based on these results, we propose a model to explain the regulation of the isoleucyl- and valyt -tRNA synthetases in which transient inhibition of the synthetase enzyme activities by threonine deaminase and 2-ketobutyrate increases the expression of ileS and valS , the structural genes for isoleucyl- and valyt -tRNA synthetase, respectively. Further, the results suggest that the hyperattenuated expression of the ilv biosynthetic operons is due to an increased rate of complex formation of valyl and isoleucyl-tRNA synthetases and the altered form of threonine deaminase of the ilvA538 mutant strain.

Regulation of synthesis of the acetohydroxy acid synthase I isoenzyme in Escherichia coli K-12☆

Abstract We studied derivatives of Escherichia coli K-12 expressing only one of the three acetohydroxy acid synthases, acetohydroxy acid synthase I (the ilvB gene product), to gain insight into the mechanism of synthesis of this isoenzyme. We found that acetohydroxy acid synthase I is regulated by a multivalent control requiring leucine and valine. In fact, a starvation of each of these two amino acids causes a derepressed synthesis of acetohydroxy acid synthase I, while a starvation of isoleucine has no effect. Derepressed synthesis of this enzyme also occurs when cells are grown in the presence of α-ketobutyrate, unless the three branched-chain amino acids are also present. Additional studies with α-aminobutyric acid, an inhibitor of valyl-tRNA Val synthesis, show that valine must be charged to tRNA to participate in this regulatory system.

The regulation of the ilvADGE operon: Evidence for positive control by threonine deaminase☆

We isolated a strain (PS187) containing a mutation in the ilvA structural gene (threonine deaminase) affecting the regulation of the isoleucine and valine biosynthetic enzymes. We studied the effect of the mutation, ilvA538, on the ability of the ilvADGE operon to respond to limitations of branched chain amino acids. We found that this regulatory mutation prevents a derepression response to limitations of branched chain amino acids. The mutation is epistatic to constitutive mutations which are deficient in co-repressor synthesis. In addition, the ilvA+ allele is dominant to ilvA538 in trans, while a cis regulatory mutation, vlr-2005, is dominant in cis. We suggest a positive regulatory role for the ilvA gene product and present evidence that the apo-dimer form of threonine deaminase is the molecular species essential for an efficient depression response of the ilvADGE operon.

A Role for Threonine Deaminase in the Regulation of α-Acetolactate Biosynthesis in Escherichia coli K12

The flow of carbon to alpha-acetolactate is Escherichia coli K12 is shown to involve the endogenous pool of alpha-ketobutyrate (alpha-KB). In vivo, the acetohydroxy acid synthase (AHAS) isoenzymes have an affinity for alpha-KB sufficiently high that alpha-acetolactate production is severely limited when alpha K-B is supplied exogenously. The ability of threonine deaminase to make alpha-KB is correlated with the synthesis of the AHAS isoenzymes. Mutations in ilvA that alter the catalytic and allosteric properties of threonine deaminase affect alpha-KB production and the expression of the AHAS isoenzymes in a direct way. The ilv A538 mutation results in a feedback-hypersensitive threonine deaminase ans slow alpha-KB and AHAS production. A spontaneous revertant of an ilvA538 strain expressing a feedback-resistant threonine deaminase produces alpha-KB and AHAS more quickly. A physiological role for the activator (valine) site on threonine deaminase is proposed and valine is shown to increase alpha-KB production in vivo. Valine can thus regulate its own biosynthetic pathway without jeopardizing the production of isoleucine. The physiological implications of the role of alpha-KB in the biosynthesis of acetolactate are discussed.

Reversion of the effects of a threonine deaminase regulatory mutant by a mutation in ilvH in Escherichiacoli K-12☆

In a strain carrying an ilvA538 mutation, the ilvGEDA operon expression is decreased (hyperattenuated) and the activity and/or expression of isoleucyl- and valyl- tRNA synthetases is decreased. We have isolated two revertants of ilvA538 owing to mutations in the ilvH gene, whose product is acetohydroxy acid synthase III. The regulatory properties of these revertants are consistent with a dual role for threonine deaminase as an effector of the ilvGEDA operon and the isoleucyl- and valyl- tRNA synthetase structural genes.