Role of serine 352 in the allosteric response of Serratia marcescens aspartokinase I-homoserine dehydrogenase I analyzed by using site-directed mutagenesis.

Aspartokinase I and homoserine dehydrogenase I (AKI-HDI) from Serratia marcescens Sr41 are encoded by the thrA gene as a single polypeptide chain. Previously, a single amino acid substitution of Ser-352 with Phe was shown to produce an AKI-HDI enzyme that is not subject to threonine-mediated feedback inhibition. To determine the role of Ser-352 in the allosteric response, the thrA gene was modified by using site-directed mutagenesis so that Ser-352 of the wild-type AKI-HDI was replaced by Ala, Arg, Asn, Gln, Glu, His, Leu, Met, Pro, Thr, Trp, Tyr, or Val. The Thr-352 and Pro-352 replacements rendered AKIs sensitive to threonine. The Tyr-352 and Asn-352 substitutions led to activation, rather than inhibition, of AKI by threonine. The other replacements conferred threonine insensitivity on AKI. The threonine sensitivity of HDI was also changed by the amino acid substitutions at Ser-352. The HDI carried by the Tyr-352 mutant AKI-HDI was activated by threonine. Single amino acid replacements at Ser-352 by Ala, Asn, Gln, His, Phe, Pro, Thr, or Tyr were introduced into truncated AKI-HDIs containing the AKI and the central regions. The AKI activity of the truncated AKI-HDI containing the first 468 amino acid residues was sensitive to threonine, and introduction of the amino acid replacements did not alter the threonine sensitivity of the AKI. Another truncated AKI-HDI containing the first 462 amino acid residues possessed threonine-resistant AKI, whereas the substitutions of Ser-352 with Ala and Pro rendered AKI sensitive to threonine. The replacement of GIn-351 with Phe activated AK1 of the truncated AKI-HDI in the presence of L-threonine. These findings suggest that Ser-352 of the central region of AKI-HDI is possibly a key residue involved with the allosteric regulation of both AKI and HDI activities.     

Cloning and characterization of the mutated threonine operon (thrA(1)5A(2)5BC) of Serratia marcescens

The entire threonine operon (thrA(1)5A(2)5BC) of Serratia marcescens TLr156, which lacks threonine-mediated feedback inhibition of both aspartokinase I (AK I) and homoserine dehydrogenase I (HD I), was cloned on a multicopy plasmid pLG339. Hybrid plasmid pSK301 carried a 6.5-kb chromosomal DNA. Several derivatives of pSK301 with Tn1000 insertions were obtained. By examining the phenotypes and the physical maps of these plasmids, we could define the loci of the thrA(1)5A(2)5, thrB, and thrC genes. The thrA(1)5A(2)5 and thrC gene products were identified by the maxicell method as proteins with Mrs of 85,000 and 43,000, respectively. The thrA(1)5A(2)5 genes encode a single polypeptide similar to the thrA1A2 genes of Escherichia coli. Plasmid pSK301 was introduced into S. marcescens T-1112, in which both AK I and HD I are produced constitutively. The resulting transformant carried five to six copies of pSK301 per chromosome and produced the AK I and HD I enzymes at three to four times higher level than control strain T-1112[pLG339]. Strain T-1112[pSK301] produced four times higher levels of threonine than strain T-1112[pLG339], yielding about 35 mg of threonine per ml of a medium containing sucrose and urea.     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

Threonine Locus of Escherichia coli K-12: Genetic Structure and Evidence for an Operon

Three genes, thrA, thrB, and thrC, were previously defined and localized in the threonine locus of Escherichia coli K-12. thrA, thrB, and thrC specify the enzymes aspartokinase I-homoserine dehydrogenase I, homoserine kinase, and threonine synthetase, respectively. A complementation analysis of the threonine cluster using derivatives of a lambda phage carrying the threonine genes (lambdadthr(c)) demonstrates that: (i) thrB and thrC each consist of a single cistron; and (ii) thrA is composed of two cistrons, thrA(1) and thrA(2), although it specifies a single polypeptide chain. thrA(1) and thrA(2) correspond to aspartokinase I and homoserine dehydrogenase I, respectively. Their relative order is established. The demonstration of polar effects of mutations (nonsense or induced by phage Mu) in thrA and thrB is taken as evidence for the existence of a thrA thrB thrC operon, transcribed in this order.     

含苏氨酸操纵子重组质粒的构建及其对大肠杆菌L-苏氨酸积累的影响

摘要：【目的】通过分子生物学手段构建重组质粒,将其转入野生型大肠杆菌W3110,分析含苏氨酸操纵子基因的质粒及质粒定点突变解除反馈抑制时,对L-苏氨酸积累的影响。【方法】以W3110染色体DNA为模板,PCR扩增苏氨酸操纵子基因,即启动子THrLp、编码前导肽基因thrL以及thrA、thrB、thrC基因,通过重叠延伸PCR的方法对thrA基因定点突变,解除苏氨酸对它的反馈抑制,构建出重组表达质粒WYE112和WYE134,5 L发酵实验测定L-苏氨酸的产量。【结果】经5 L发酵罐发酵产酸实验,W3110的L-苏氨酸产量为0.036 ± 0.004 g/L,携带含苏氨酸操纵子质粒的W3110菌株L-苏氨酸产量为2.590 ± 0.115 g/L,质粒上thrA解除反馈抑制后,L-苏氨酸的产量增加到9.223 ± 1.279 g/L。【结论】过表达苏氨酸操纵子基因可以使L-苏氨酸积累,进一步解除thrA基因的反馈抑制,可以增强L-苏氨酸积累的效果,为L-苏氨酸工程菌改造的进一步研究奠定了基础。     

Mechanism of control of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase by threonine

The regulatory domain of the bifunctional threonine-sensitive aspartate kinase homoserine dehydrogenase contains two homologous subdomains defined by a common loop-alpha helix-loop-beta strand-loop-beta strand motif. This motif is homologous with that found in the two subdomains of the biosynthetic threonine-deaminase regulatory domain. Comparisons of the primary and secondary structures of the two enzymes allowed us to predict the location and identity of the amino acid residues potentially involved in two threonine-binding sites of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase. These amino acids were then mutated and activity measurements were carried out to test this hypothesis. Steady-state kinetic experiments on the wild-type and mutant enzymes demonstrated that each regulatory domain of the monomers of aspartate kinase-homoserine dehydrogenase possesses two nonequivalent threonine-binding sites constituted in part by Gln(443) and Gln(524). Our results also demonstrated that threonine interaction with Gln(443) leads to inhibition of aspartate kinase activity and facilitates the binding of a second threonine on Gln(524). Interaction of this second threonine with Gln(524) leads to inhibition of homoserine dehydrogenase activity.     

Transductional construction of a threonine-producing strain of Serratia marcescens.

A threonine-producing strain of Serratia marcescens Sr41 was constructed according to the following process. Thr- strain E-60 was derived from strain HNr59 having constitutive levels of threonine-sensitive aspartokinase and homoserine dehydrogenase. Thr+ transductant T-570 was constructed from strain E-60 and phage grown on strain HNr21 having feedback-resistant threonine-sensitive aspartokinase and homoserine dehydrogenase. This transductant lacked both feedback inhibition and repression for the two enzymes. Thr- strain N-11 was derived from strain AECr174 lacking feedback inhibition and repression of lysine-sensitive aspartokinase. Subsequently, the threonine region of strain T-570 was transduced into strain N-11. One of the THR+ transductants, strain T-693, produced markedly high levels of the two aspartokinases and homoserine dehydrogenase, which were insensitive to feedback inhibition. This strain produced about 25 mg of threonine per ml in the medium containing sucrose and urea.     

Quantitative estimates of the distribution of homoserine dehydrogenase isozymes in maize tissues

The low molecular weight threonine-resistant (class I) and the higher molecular weight threonine-sensitive (class II/III) isozymes of homoserine dehydrogenase (EC 1.1.1.3) isolated from Zea mays L. were shown to differ in stability during incubations in the presence of urea. Class II/III was inactivated by urea in a time- and concentration-dependent manner, with complete inactivation occurring within 24 hours at 5 degrees C in 4.0 m urea. Under identical conditions, neither the activity nor the properties of class I were affected. Therefore, it was possible to estimate the amounts and properties of both maize isozymes in crude mixtures by measurements of enzyme activity before and after treatment with urea.The relative amounts of the two isozymes proved to be tissue-specific. When shoots of etiolated seedlings were extracted under optimum conditions, the resultant preparations contained about 16% class I and 84% class II/III. This distribution of isozymes, as well as the regulatory properties of class II/III, were constant during growth of the seedlings between 4 and 13 days. Enzyme preparations isolated from shoots of light-grown plants contained higher proportions of class I. The two isozymes were not uniformly distributed within leaves, as the basal meristematic region contained high levels of II/III and small amounts of I. During leaf maturation, the amount of II/III declined while the level of I remained constant or increased slightly. As a result, nearly half of the enzyme extracted from leaf tips was class I. The synthesis of specific members of the aspartate family of amino acids might be expected to differ when the ratio of threonine-sensitive to threonine-resistant homoserine dehydrogenase is altered. However, additional information on the subcellular localization and the catalytic characteristics of the two enzymes is required for evaluation of this possibility.     

Transductional construction of a threonine-producing strain of Serratia marcescens.

A threonine-producing strain of Serratia marcescens Sr41 was constructed according to the following process. Thr- strain E-60 was derived from strain HNr59 having constitutive levels of threonine-sensitive aspartokinase and homoserine dehydrogenase. Thr+ transductant T-570 was constructed from strain E-60 and phage grown on strain HNr21 having feedback-resistant threonine-sensitive aspartokinase and homoserine dehydrogenase. This transductant lacked both feedback inhibition and repression for the two enzymes. Thr- strain N-11 was derived from strain AECr174 lacking feedback inhibition and repression of lysine-sensitive aspartokinase. Subsequently, the threonine region of strain T-570 was transduced into strain N-11. One of the THR+ transductants, strain T-693, produced markedly high levels of the two aspartokinases and homoserine dehydrogenase, which were insensitive to feedback inhibition. This strain produced about 25 mg of threonine per ml in the medium containing sucrose and urea.     

Yeast homoserine kinase. Characteristics of the corresponding gene, THR1, and the purified enzyme, and evolutionary relationships with other enzymes of threonine metabolism

THR1, the gene from Saccharomyces cerevisiae, encoding homoserine kinase, one of the threonine biosynthetic enzymes, has been cloned by complementation. The nucleotide sequence of a 3.1-kb region carrying this gene reveals an open reading frame of 356 codons, corresponding to about 40 kDa for the encoded protein. The presence of three canonical GCN4 regulatory sequences in the upstream flanking region suggests that the expression of THR1 is under the general amino acid control. In parallel, the enzyme was purified by four consecutive column chromatographies, monitoring homoserine kinase activity. In SDS gel electrophoresis, homoserine kinase migrates like a 40-kDa protein; the native enzyme appears to be a homodimer. The sequence of the first 15 NH2-terminal amino acids, as determined by automated Edman degradation, is in accordance with the amino acid sequence deduced from the nucleotide sequence. Computer-assisted comparison of the yeast enzyme with the corresponding activities from bacterial sources showed that several segments among these proteins are highly conserved. Furthermore, the observed homology patterns suggest that the ancestral sequences might have been composed from separate (functional) domains. A block of very similar amino acids is found in the homoserine kinases towards the carboxy terminus that is also present in many other proteins involved in threonine (or serine) metabolism; this motif, therefore, may represent the binding site for the hydroxyamino acids. Limited similarity was detected between a motif conserved among the homoserine kinases and consensus sequences found in other mono- or dinucleotide-binding proteins.     

Aspartokinase of Lemna paucicostata Hegelm. 6746

A sensitive and specific method was developed for assay of aspartokinase (EC 2.7.2.4) in crude extracts of Lemna paucicostata. Lysine inhibited approximately 93%, and threonine approximately 6%; together, these amino acids inhibited 99%. Inhibition by lysine was synergistically increased by S-adenosylmethionine, which by itself had no effect on activity. Essentially complete inhibition of threonine-resistant activity was obtained with lysine, and of lysine-resistant activity with threonine. Inhibition by lysine and threonine was additive, with no indication of concerted inhibition. Aspartate concentration had no effect on the relative proportions of lysine- and threonine-sensitive activities. Aspartokinase activity was in large excess of that reported by other workers, the maximum capacity (V_max) far exceeding the in vivo requirements. Estimations of rates of aspartokinase in vivo suggest that the step catalyzed by this enzyme may not be the overall `rate-limiting' one for entry of 4-carbon units into the aspartate family of amino acids, and that feedback inhibition of this enzyme by lysine and threonine may not be a major factor in regulating flux through this step.     

Participation of lysine-sensitive aspartokinase in threonine production by S-2-aminoethyl cysteine-resistant mutants of Serratia marcescens.

S-2-Aminoethyl cysteine (AEC) reduced both growth rate and final growth level of Serratia marcescens Sr41. The growth inhibition was completely reversed by lysine. AEC inhibited the activity of lysine-sensitive aspartokinase to a lesser extent than lysine. The AEC addition to the medium lowered not only the level of lysine-sensite aspartokinase but also those of homoserine dehydrogenase and threonine deaminase, whereas lysine repressed the aspartokinase alone. To select mutations releasing lysine-sensitive aspartokinase from feedback controls, AEC-resistant colonies were isolated from strains HNr31 and HNr53, both of which were previously found to excrete threonine on the minimal plates but not on the plates containing excess lysine. Two of 280 resistant colonies excreted large amounts of threonine. Strains AECr174 and AECr301, derived from strains HNr31 and HNr53, respectively, lacked both feedback inhibition and repression of lysine-sensitive aspartokinase. These strains produced about 7 mg of threonine per ml in the medium containing glucose and urea.     

Participation of lysine-sensitive aspartokinase in threonine production by S-2-aminoethyl cysteine-resistant mutants of Serratia marcescens.

S-2-Aminoethyl cysteine (AEC) reduced both growth rate and final growth level of Serratia marcescens Sr41. The growth inhibition was completely reversed by lysine. AEC inhibited the activity of lysine-sensitive aspartokinase to a lesser extent than lysine. The AEC addition to the medium lowered not only the level of lysine-sensite aspartokinase but also those of homoserine dehydrogenase and threonine deaminase, whereas lysine repressed the aspartokinase alone. To select mutations releasing lysine-sensitive aspartokinase from feedback controls, AEC-resistant colonies were isolated from strains HNr31 and HNr53, both of which were previously found to excrete threonine on the minimal plates but not on the plates containing excess lysine. Two of 280 resistant colonies excreted large amounts of threonine. Strains AECr174 and AECr301, derived from strains HNr31 and HNr53, respectively, lacked both feedback inhibition and repression of lysine-sensitive aspartokinase. These strains produced about 7 mg of threonine per ml in the medium containing glucose and urea.     

Influence of threonine exporters on threonine production in Escherichia coli

Threonine production in Escherichia coli threonine producer strains is enhanced by overexpression of the E. coli rhtB and rhtC genes or by heterologous overexpression of the gene encoding the Corynebacterium glutamicum threonine excretion carrier, thrE. Both E. coli genes give rise to a threonine-resistant phenotype when overexpressed, and they decrease the accumulation of radioactive metabolites derived from [(14)C] L-threonine. The evidence presented supports the conclusion that both RhtB and RhtC catalyze efflux of L-threonine and other structurally related neutral amino acids, but that the specificities of these two carriers differ substantially.     

Threonine-insensitive Homoserine Dehydrogenase from Soybean: GENOMIC ORGANIZATION, KINETIC MECHANISM, AND IN VIVO ACTIVITY*

Aspartate kinase (AK) and homoserine dehydrogenase (HSD) function as key regulatory enzymes at branch points in the aspartate amino acid pathway and are feedback-inhibited by threonine. In plants the biochemical features of AK and bifunctional AK-HSD enzymes have been characterized, but the molecular properties of the monofunctional HSD remain unexamined. To investigate the role of HSD, we have cloned the cDNA and gene encoding the monofunctional HSD (GmHSD) from soybean. Using heterologously expressed and purified GmHSD, initial velocity and product inhibition studies support an ordered bi bi kinetic mechanism in which nicotinamide cofactor binds first and leaves last in the reaction sequence. Threonine inhibition of GmHSD occurs at concentrations (K_i = 160–240 mm) more than 1000-fold above physiological levels. This is in contrast to the two AK-HSD isoforms in soybean that are sensitive to threonine inhibition (K_i∼150 μm). In addition, GmHSD is not inhibited by other aspartate-derived amino acids. The ratio of threonine-resistant to threonine-sensitive HSD activity in soybean tissues varies and likely reflects different demands for amino acid biosynthesis. This is the first cloning and detailed biochemical characterization of a monofunctional feedback-insensitive HSD from any plant. Threonine-resistant HSD offers a useful biotechnology tool for manipulating the aspartate amino acid pathway to increase threonine and methionine production in plants for improved nutritional content.     

Regulatory isoenzymes of aspartate kinase and the control of lysine and threonine biosynthesis in carrot cell suspension culture

Aspartate kinase (EC 2.7.2.4) from carrot (Daucus carota L.) cell suspension culture has been partially resolved into lysine-sensitive and threonine-sensitive components by gel filtration chromatography. The yield of lysine-sensitive aspartate kinase changed independently of the yield of the threonine-sensitive activity during the 4-week growth cycle of the culture, and this provides additional evidence for the existence of two independently regulated isoenzymes. Exogenously supplied lysine and threonine specifically inhibited the in vivo formation of lysine and threonine, respectively, from radioactive acetate.