Analysis of the functional domains of biosynthetic threonine deaminase by comparison of the amino acid sequences of three wild-type alleles to the amino acid sequence of biodegradative threonine deaminase

Taillon, Bruce E.; Little, R. A.; Lawther, Robert P.

doi:10.1016/0378-1119(88)90528-8

Cited by 30 publications

(25 citation statements)

References 29 publications

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“…3, the 145 clones isolated in this study are localized predominantly in the amino-terminal half of threonine deaminase, and no mutations that affected catalysis were identified in the carboxy-terminal third of the polypeptide chain. This distribution is consistent with several suggestions that the enzyme is organized functionally into an amino-terminal catalytic domain and a carboxy-terminal regulatory domain (1,29,35).…”

Section: Discussionsupporting

confidence: 92%

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An efficient approach to identify ilvA mutations reveals an amino-terminal catalytic domain in biosynthetic threonine deaminase from Escherichia coli

Fisher

Eisenstein

1993

J Bacteriol

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High-level expression of the regulatory enzyme threonine deaminase in Escherichia coli strains grown on minimal medium that are deficient in the activities of enzymes needed for branched-chain amino acid biosynthesis result in growth inhibition, possibly because of the accumulation of toxic levels of a-ketobutyrate, the product of the committed step in isoleucine biosynthesis. This condition affords a means for selecting genetic variants of threonine deaminase that are deficient in catalysis by suppression of growth inhibition.Strains harboring mutations in ilvA that decreased the catalytic activity of threonine deaminase were found to grow more rapidly than isogenic strains containing wild-type ilvA. Modification of the ilvA gene to introduce additional unique, evenly spaced restriction enzyme sites facilitated the identification of suppressor mutations by enabling small DNA fragments to be subcloned for sequencing. The 10 mutations identified in ilvA code for enzymes with significantly reduced activity relative to that of wild-type threonine deaminase. Values for their specific activities range from 40%o of that displayed by wild-type enzyme to complete inactivation as evidenced by failure to complement an ilvA deletion strain to isoleucine prototrophy. Moreover, some mutant enzymes showed altered allosteric properties with respect to valine activation and isoleucine inhibition. The location of the 10 mutations in the 5' two-thirds of the ilvA gene is consistent with suggestions that threonine deaminase is organized functionally with an amino-terminal domain that is involved in catalysis and a carboxy-terminal domain that is important for regulation.The regulatory enzyme threonine deaminase [threonine dehydratase; L-threonine hydro-lyase (deaminating); EC 4.2.1. 16] from Escherichia coli exerts enzymological control at the committed step of branched-chain amino acid biosynthesis as evidenced by a sigmoidal dependence of its initial velocity on threonine concentration (8,34). In addition, the enzyme is allosterically regulated by the heterotropic effectors isoleucine and valine, which either inhibit or activate, respectively, the pyridoxal phosphate-dependent catalytic reaction at intermediate substrate concentrations. A molecular description of the structural and energetic changes that occur at the active sites when regulatory ligands bind to the effector sites is a key issue in the analysis of the cooperative behavior of threonine deaminase. A powerful strategy in studies focusing on the mechanism of communication between effector sites and active sites in other allosteric systems involves correlating the structural locations of genetic variants with the functional alterations that result in the mutant proteins (16, 32). At present, however, there is no high-resolution structure of threonine deaminase, and hence there is little information about the importance of specific amino acids for catalysis, feedback regulation, and the allosteric transition. In an effort to identify residues in the enzyme that are inv...

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Section: Discussionsupporting

confidence: 92%

“…The locations of these substitutions in the ilvA gene are consistent with suggestions that threonine deaminase is functionally organized with an amino-terminal domain that is responsible for catalysis (29,35).…”

supporting

confidence: 84%

An efficient approach to identify ilvA mutations reveals an amino-terminal catalytic domain in biosynthetic threonine deaminase from Escherichia coli

Fisher

Eisenstein

1993

J Bacteriol

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“…However, when the carboxy-terminal portion of the C. glutamicum sequence is considered, the situation is entirely different. A corresponding sequence is not present in tdc; this sequence is therefore suggested to be of relevance for allosteric regulation in the biosynthetic enzyme (ilvA) (45). In this carboxy-terminal portion of the C. glutamicum sequence, no distinct blocks of identical amino acids are conserved, as in the amino-terminal portion.…”

Section: Resultsmentioning

confidence: 99%

“…Possibly, the number of binding sites, pyridoxal 5'-phosphate content, and the oligomeric state are involved, as shown for the enzyme of S. typhimurium (19,23). There are several indications from mutant enzymes of E. coli and a homology comparison of the biosynthetic threonine dehydratase (ilvA) with its catabolic counterpart (tdc) that the carboxy-terminal portion is involved in allosteric regulation of the enzyme (10,15,45).…”

mentioning

confidence: 99%

Functional and structural analyses of threonine dehydratase from Corynebacterium glutamicum

1992

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Threonine dehydratase activity is an important element in the flux control of isoleucine biosynthesis. The enzyme of Corynebacterium gluamicum demonstrates a marked sigmoidal dependence of initial velocity on the threonine concentration, a dependence that is consistent with substrate-promoted conversion of the enzyme from a low-activity to a high-activity conformation. In the presence of the negative aliosteric effector isoleucine, the K.45 increased from 21 to 78 mM and the cooperativity, as expressed by the Hill coefficient increased from 2.4 to 3.7. Valine promoted opposite effects: the K0.5 was reduced to 12 mM, and the enzyme exhibited almost no cooperativity. Sequence determination of the C. ghutamicum gene for this enzyme revealed an open reading frame coding for a polypeptide of 436 amino acids. From this information and the molecular weight determination of the native enzyme, it follows that the dehydratase is a tetramer with a total mass of 186,396 daltons. Comparison of the deduced polypeptide sequence with the sequences of known threonine dehydratases revealed surprising differences from the C. glutamicum enzyme in the carboxy-terminal portion. This portion is greatly reduced in size, and a large gap of 95 amino acids must be introduced to achieve homology.

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“…2). This threonine dehydratase region was previously designated as the C3 domain and is proposed to be involved in the catalysis of the enzyme (18). Furthermore, there is similarity between this region and a specific region of several eucaryotic serine or threonine kinases (Fig.…”

mentioning

confidence: 96%

Analysis of a Corynebacterium glutamicum hom gene coding for a feedback-resistant homoserine dehydrogenase

1991

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From a Corynebacterium glutamicum mutant possessing a homoserine dehydrogenase resistant to feedback inhibition by L-threonine, the corresponding gene (homFBR) was analyzed and compared with the wild-type hom gene. DNA fragment exchange experiments between both genes showed that a 0.23-kb region close to the 3' terminus of homFBR was responsible for deregulation. Nucleotide sequence analysis revealed a single transition from G to A in homFBR leading to replacement of glycine-378 by glutamate in the mutant homoserine dehydrogenase.In coryneform bacteria, L-threonine biosynthesis starting with aspartate consists of five enzymatic steps, the initial two being common to L-threonine and L-lysine synthesis. Carbon flow towards L-threonine is regulated by the third enzyme, homoserine dehydrogenase (HDH), which is inhibited by L-threonine (9, 10). Moreover, synthesis of HDH is weakly repressed by methionine (11). Mutants of Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum, possessing an HDH insensitive to inhibition by L-threonine, show increased L-threonine formation and are therefore used in industrial L-threonine production (6, 16).The C. glutamicum hom gene coding for the wild-type HDH was previously isolated and analyzed by Peoples et al. (14). We present here the analysis of a C. glutamicum hom gene (homFBR) coding for a feedback-resistant HDH. The homFBR gene was derived from C. glutamicum mutant strain DM 368-3, which was obtained by N-methyl-N'-nitro-Nnitrosoguanidine mutagenesis and subsequent selection on medium containing the threonine analog 2-amino-3-hydroxyvalerate (9 mg/ml).The homFBR gene from C. glutamicum DM 368-3 (Degussa AG) and the wild-type hom gene from C. glutamicum ATCC 13032 were previously isolated as 3.6-kb Sall-generated chromosomal fragments (3). For the following experiments, hom and homFBR were subcloned as 1.7-kb SmaI-XmnI fragments in the Escherichia coliIC. glutamicum shuttle vector pJC1 (2). After transformation of pJC1-hom and pJC1-homFBR into C. glutamicum DM 368-3 by electroporation (8), both constructs conferred about 12-fold overexpression of HDH (Fig. 1). As expected, pJC1-hom expressed an HDH that was completely inhibited by 2 mM L-threonine, whereas pJC1-homFBR expressed an HDH that showed no inhibition by L-threonine up to 25 mM.To localize the region in homFBR responsible for deregulation of HDH, we exchanged fragments between pJC1-hom and pJC1-homFBR, transformed the hybrid constructs into C. glutamicum DM 368-3, and tested the constructs for the expression of a regulated or deregulated HDH (Fig. 1). In pJC1-hom, a 0.5-kb PvuII-BamHI fragment containing the terminal 412 bp of hom was replaced by the corresponding fragment from pJC1-homFBR, resulting in the hybrid con-* Corresponding author. struct pJC1-hom/rell. This plasmid expressed an HDH with insensitivity towards L-threonine equal to that of the mutant HDH (Fig. 1). In an analogous experiment, the 0.5-kb PvuII-BamHI fragment from pJC1-homFBR was replaced by the corresponding region...

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Analysis of the functional domains of biosynthetic threonine deaminase by comparison of the amino acid sequences of three wild-type alleles to the amino acid sequence of biodegradative threonine deaminase

Cited by 30 publications

References 29 publications

An efficient approach to identify ilvA mutations reveals an amino-terminal catalytic domain in biosynthetic threonine deaminase from Escherichia coli

An efficient approach to identify ilvA mutations reveals an amino-terminal catalytic domain in biosynthetic threonine deaminase from Escherichia coli

Functional and structural analyses of threonine dehydratase from Corynebacterium glutamicum

Analysis of a Corynebacterium glutamicum hom gene coding for a feedback-resistant homoserine dehydrogenase

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