Threonine metabolism byPseudomonas aeruginosa

83 strains ofPseudomonas aeruginosa were unable to utilizel-threonine as carbon-energy source, although this compound served as sole nitrogen source. Auxotrophs ofP. aeruginosa 9-D2 that requiredl-serine or glycine for growth could grow in the presence ofl-threonine. Extracts ofP. aeruginosa 9-D2 grown in the presence ofl-threonine contained threonine dehydrogenase and alpha-amino beta-ketobutyrate: CoA ligase activities; threonine aldolase was not detectable. Cells grown in the absence ofl-threonine produced no detectable threonine dehydrogenase.l-Leucine neither stimulated nor repressed threonine dehydrogenase levels. Glycine, and to a lesser extentl-serine, repressedl-threonine-mediated threonine dehydrogenase synthesis. A mutant of strain 9-D2 was isolated that could utilizel-threonine as sole carbon-energy source. This strain produced elevated levels of threonine dehydrogenase, but only slightly higher levels of alpha-amino beta-ketobutyrate: CoA ligase activities.     

Threonine metabolism in Japanese quail liver

Summary. In general, threonine is metabolized by reaction catalyzed by threonine-3-dehydrogenase (TDH), threonine dehydratase (TH) or threonine aldolase (TA). The activities of these three enzymes were compared in the liver of Japanese quails and rats. The animals were fed a standard or threonine rich-diet, or fasted for 3 days. The specific activity of TDH in the liver from quail fed a standard diet was 11 times higher than that in the liver from rats fed a standard diet. The TDH activities in the livers of the fasting and 5% threonine-rich diet groups of quail were 3 and 2 times higher than those in the livers from quail fed the standard diet, respectively. The TH activity in the liver of rats fed a standard diet was 14 times higher than that in the liver of quail fed a standard diet. The TH activity in the rat liver after fasting was 2.3 times higher than that of the standard diet control. The activity of TA in the livers of rat and quail were so low that its role in threonine metabolism in both animals seemed to be negligible. These results suggest that threonine is a ketogenic amino acid in the quail liver, while it is a glucogenic in the rat liver.     

Nutrition of Threonine

The threonine content in blood and urine increased and threonine decomposition ability in liver decreased by feeding lower level of lysine, whereas threonine content in blood and urine decreased and the ability of liver increased gradually with increasing lysine content in diet. These phenomena were owing to the increase of threonine dehydratase activity of liver, which was measured from produced α-ketobutyric acid amount, by excess administration of lysine. The phenomena that threonine content in urine decreased and threonine decomposition ability of liver increased with increasing threonine content in diet when adequate amount of lysine was fed, were also ascribed to the increase of the dehydratase activity.

One m mole of threonine was incubated with liver homogenate in presence of PALP*** at pH 8.2 for 20 and 30 min and α-ketobutyric acid produced was introduced to its 2,4-dinitrophenylhydrazone, which was chromatographed on silica-gel thin-layer plate and determined spectrophotometrically at 395 mμ under N,N-dimethylformamide.

Other enzyme systems relating to threonine catabolism were also investigated, including threonine aldolase, threonine dehydrogenase and ornithine transaminase, showing no significant changes in activities by excess administration of lysine and/or threonine.     

Nutrition of Threonine

By administering 2 mg/day of cortisone acetate to adrenalectomized rats, the hepatic threonine dehydratase activity of these rats increased 5 times as much as that of the control. By administering 5 IU/day of ACTH to hypophysectomized rats, both the hepatic threonine dehydratase activity and the adrenal glucose-6-phosphate dehydrogenase activity increased 3 times and 7 times as much as that of the control group, respectively. The effects of excess feeding of lysine or threonine on the increase of the dehydratase activity by the adminitration of cortisone to the adrenalectomized rats and the administration of ACTH to the hypophysetomized rats were negative. When the intact rats were fed on lysine and/or threonine excess diet, the amount of glucocorticoid secretion as measured by the adrenal glucose-6-phosphate dehydrogenase activity increased and the hepatic threonine dehydratase activity increased accordingly. A linear relationship was found between these two activities and no significant deviation from the relationship due to the difference in diet composition was observed. A mechanism was proposed, based on these results, explaining the fact that the hepatic threonine dehydratase activity increased when rats were fed on lysine or threonine excess diet.     

Nutrition of Threonine

Among the threonine dehydratase activities in liver of rat fed on basal diet (B-group), lysine added diet (L-group) and threonine added diet (T-group), a following relationship was found: A_B?A_LT, where A_B, A_L and A_T were the activities of B-, L-and T-group, respectively. The types of the activity increase in L- or T-group were investigated by examining the possibility of the cause—the existence of activators in L- or T-group (I), the existence of inhibitors in B-group (II), the activation of the latent enzyme in L- or T-group (III), and the induction of the dehydratase by biosynthesis in L- or T-group (IV). Addition of various amounts of liver homogenate of L- or T-group to a given amount of that of B-group gave a result as would be obtained in cases where neither activators nor inhibitors existed in the liver (I and II). No correlationship was found between the activity and the preincubation time, which denied the presence of the latent enzyme which would easily change into the active form by preincubation (III). Actinomycin D administered to rats inhibited the increase of the dehydratase activity of L- or T-group by about 50% (IV). On the other hand, preliminary experiments using hypophysectomized or adrenal-ectomizecl rats showed the results of A_в?A_L?A_т. Both results may suggest the possibility that the increase of the dehydratase activity is ascribed to the induction of this enzyme through biosynthesis, and perhaps through endocrine systems.     

Threonine Aldolase Overexpression plus Threonine Supplementation Enhanced Riboflavin Production in Ashbya gossypii

Riboflavin production in the filamentous fungus Ashbya gossypii is limited by glycine, an early precursor required for purine synthesis. We report an improvement of riboflavin production in this fungus by overexpression of the glycine biosynthetic enzyme threonine aldolase. The GLY1 gene encoding the threonine aldolase of A. gossypii was isolated by heterologous complementation of the glycine-auxotrophic Saccharomyces cerevisiae strain YM13 with a genomic library from A. gossypii. The deduced amino acid sequence of GLY1 showed 88% similarity to threonine aldolase from S. cerevisiae. In the presence of the GLY1 gene, 25 mU of threonine aldolase specific activity mg⁻¹ was detectable in crude extracts of S. cerevisiae YM13. Disruption of GLY1 led to a complete loss of threonine aldolase activity in A. gossypii crude extracts, but growth of and riboflavin production by the knockout mutant were not affected. This indicated a minor role of the enzyme in glycine biosynthesis of A. gossypii. However, overexpression of GLY1 under the control of the constitutive TEF promoter and terminator led to a 10-fold increase of threonine aldolase specific activity in crude extracts along with a 9-fold increase of riboflavin production when the medium was supplemented with threonine. This strong enhancement, which could not be achieved by supplementation with glycine alone, was attributed to an almost quantitative uptake of threonine and its intracellular conversion into glycine. This became evident by a subsequent partial efflux of the glycine formed.     

苏氨酸醛缩酶的研究进展

苏氨酸醛缩酶(TAs)以磷酸吡哆醛为辅酶,催化不同的醛与α-氨基酸发生醇醛缩合反应,形成具有2个手性中心的β-羟基-α-氨基酸。TAs在不对称催化过程中可以控制产物α-碳位的立体构型,而对β-碳位的立体构型控制相对较弱。增强TAs在β-碳位的立体选择性是近年来研究TAs不对称催化的热点。本文重点阐述了TAs的结构与作用机制、定向进化,以及在生物催化合成中的应用,对TAs的研究与开发进行了展望。     

Threonine degradation by Serratia marcescens.

The wild strain of Serratia marcescens rapidly degraded threonine and formed aminoacetone in a medium containing glucose and urea. Extracts of this strain showed high threonine dehydrogenase and "biosynthetic" threonine deaminase activities, but no threonine aldolase activity. Threonine dehydrogenase-deficient strain Mu-910 was selected among mutants unable to grow on threonine as the carbon source. This strain did not form aminoacetone from threonine, but it slowly degraded threonine. Strain D-60, deficient in both threonine dehydrogenase and threonine deaminase, was derived from strain Mu-910 and barely degraded threonine. A glycine-requiring strain derived from the wild strain grew in minimal medium containing threonine as the glycine source, whereas a glycine-requiring strain derived from strain Mu-910 did not grow. This indicates that threonine dehydrogenase participates in glycine formation from threonine (via alpha-amino-beta-ketobutyrate) as well as in threonine degradation to aminoacetone.     

Threonine inhibition of the aspartokinase--homoserine dehydrogenase I of Escherichia coli. Threonine binding studies.

Both activities of the aspartokinase--homoserine I (AK-HSD) of Escherichia coli are inhibited by threonine. Careful threonine binding studies have now been done which have allowed us to distinguish the various effects of threonine on the enzyme. The ultrafiltration technique of H. Paulus ((1969) Anal. Biochem. 32, 101) for measuring ligand binding was shown to be comparable with equilibrium dialysis techniques. Reduction in error by utilization of this procedure enabled us to obtain evidence for two different sets of threonine sites by direct binding studies. The binding data were mathematically consistent with two independent classes of threonine sites, each of which contained four sites per tetramer and had a Hill coefficient of about 2.3--2.5. KD for the second set of sites was five- to tenfold greater than the high affinity sites, depending upon conditions. The data now suggest that the sequential model for site--site interactions adequately describes the cooperativity of threonine binding to the high affinity set of sites.     

Inhibition of Threonine Dehydratase Is Herbicidal

Threonine dehydratase, the first enzyme in isoleucine biosynthesis, catalyzes deamination and dehydration of threonine to produce 2-ketobutyrate and ammonia. An antimetabolite, 2-(1-cyclohexen-3(R)-yl)-S-glycine (CHG), inhibits the plant enzyme. CHG inhibits the growth of Black Mexican Sweet corn (Zea mays) cells and of Arabidopsis thaliana plants. The herbicidal effects of CHG can be reversed by 2-ketobutyrate, other intermediates of isoleucine biosynthesis, and by isoleucine itself. These results suggest that the herbicidal effects observed with CHG are a consequence of inhibition of threonine dehydratase. The enzyme could be a potential target site for an herbicide screening program.     

Biosynthetic Threonine Deaminase from Proteus morganii

Biosynthetic threonine deaminase was purified to an apparent homogeneous state from the cell extract of Proteus morganii, with an overall yield of 7.5%. The enzyme had a s⁰_20,w of 10.0 S, and the molecular weight was calculated to be approximately, 228,000. The molecular weight of a subunit of the enzyme was estimated to be 58,000 by sodium dodecyl sulfate gel electrophoresis. The enzyme seemed to have a tetrameric structure consisting of identical subunits. The enzyme had a marked yellow color with an absorption maximum at 415 nm and contained 2 mol of pyridoxal 5′-phosphate per mol. The threonine deaminase catalyzed the deamination of l-threonine, l-serine, l-cysteine and β-chloro-l-alanine. Km values for l-threonine and l-serine were 3.2 and 7.1 mm, respectively. The enzyme was not activated by AMP, ADP and ATP, but was inhibited by l-isoleucine. The Ki for l-isoleucine was 1.17 mm, and the inhibition was not recovered by l-valine. Treatment with mercuric chloride effectively protected the enzyme from inhibition by l-isoleucine.     

Mixed Crystals of Threonine and Allothreonine

Study of the soluble proteins of sweet potato (Ipomoea batatas L. cv. Norin 1) roots showed that the major protein had an apparent molecular weight of 25,000, and accounted for 60 ~ 70% of the total soluble protein extracted from fresh tissue. The 25-kDa protein exists in two forms, which can be resolved into two bands by nondenaturing polyacrylamide gel electrophoresis. Immunodiffusion and crossed immunoelectrophoresis showed that these forms are immunologically identical. This protein was identified as the antigenic component A of sweet potato root.¹⁾ It was degraded to proteins of lower molecular weight (9,500 to 20,000) if the tissue was cut or infected by Ceratocystis fimbriata. As almost none of this 25-kDa protein was detected in roots stored for one year at 10 ~ 12°C, it is probably the storage protein of these roots. Another major protein was identified as β-amylase by immunodiffusion and immunoelectrophoresis. The amount of β-amylase did not change appreciably after cutting or infection, but it was present in only trace amounts in the roots stored for one year, Cutting, infection, or storage of root tissue resulted in the production of new isozymes of peroxidase, acid phosphatase, and esterase. Increases in some other proteins in cut and in diseased tissues were detected by gel electrophoresis.