Jean-Claude Patte

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli: I. Evidence that the two activities are carried by a single protein

In Escherichia coli K12, 1. 1. A single mutation can lead to the concomitant modification or to the concomitant loss of the two activities, threonine-sensitive β-aspartokinase (ATP: L-aspartate 4-phosphotransferase, EC 2.7.2.4) and threonine-sensitive homoserine dehydrogenase (L-homoserine: NADP+ oxidoreductase, EC 1.1.1.3). 2. 2. The two activities cannot be separated and the ratio of the specific activities remains constant throughout a 600-fold purification. 3. 3. The substrates of one of the activities are inhibitors of the otther activity. The observed inhibitions are specific. 4. 4. The threonine-sensitive aspartokinase is protected against thermal inactivation by NADPH, a substrate of the homoserine dehydrogenase. 5. 5. The conclusion is drawn that the two activities under study are carried by a single protein molecule. The apparent molecular mass of this complex protein is changed in some mutants. There is no apparent correlation between the apparent molecular mass and the cooperativity of inhibitor molecules. 6. 6. The significance of these findings is discussed in terms of metabolic regulation and intracellular topology.

Rétro-inhibition et répression de l'homosérine déshydrogénase d'Escherichia coli

Abstract The homoserine dehydrogenase of Escherichia coli K12 is under the control of the two types of known regulatory negative feedback mechanisms: repression and end-product inhibition. The regulatory agent is l -threonine. End-product inhibition by l -threonine is highly specific and strictly non-competitive. The inhibition site of the enzyme can be selectively destroyed by heat. The stability of the enzyme and of its capacity to be inhibited have been studied. The sedimentation constant of the inhibitable enzyme is higher than that of the enzyme having lost its inhibition site. The synthesis of E. coli homoserine dehydrogenase is greatly and specifically repressed by l -threonine in the culture medium. The genetic control of the synthesis of the aspartic acid family of amino acids is discussed.

Regulation by methionine of the synthesis of third aspartokinase and of a second homoserine dehydrohenase in Escherichia coli K 12

Abstract 1. 1. Evidence is presented that, in addition to the lysine-sensitive aspartokinase III and to the threonine-sensitive association of aspartokinase I and homoserine dehydrogenase I, there exist in Escherichia coli K 12 two heretofore undetected enzymes: aspartokinase II and homoserine dehydrogenase II. The synthesis of these two enzymes is under repressive control by methionine. 2. 2. Some of their properties are described: it should be noted that their activity is not end-product regulated. 3. 3. A constitutive mutant for the two novel activities has been isolated. 4. 4. In E. coli B, homoserine dehydrogenase II is absent. 5. 5. The overall features of repression and end-product inhibition in branched biosynthetic pathways are discussed.

A direct sulfhydrylation pathway is used for methionine biosynthesis in Pseudomonas aeruginosa.

The relationship between genes and enzymes in the methionine biosynthetic pathway has been studied in Pseudomonas aeruginosa. The first step is catalysed by an O-succinylhomoserine synthase, the product of the metA gene mapped at 20 min on the chromosome. The second step is achieved by direct sulfhydrylation, involving the enzyme encoded by a metZ gene that we have identified and sequenced, located at 40 min. Thus Pseudomonas appears to be the only organism so far described that uses O-succinylhomoserine as substrate for a direct sulfhydrylation. As in yeast, the two transsulfuration pathways between cysteine and homocysteine, with cystathionine as an intermediate, probably exist in parallel in this organism.

The torYZ (yecK bisZ) Operon Encodes a Third Respiratory Trimethylamine N-Oxide Reductase in Escherichia coli

The bisZ gene of Escherichia coli was previously described as encoding a minor biotin sulfoxide (BSO) reductase in addition to the main cytoplasmic BSO reductase, BisC. In this study, bisZ has been renamed torZ based on the findings that (i) the torZ gene product, TorZ, is able to reduce trimethylamine N-oxide (TMAO) more efficiently than BSO; (ii) although TorZ is more homologous to BisC than to the TMAO reductase TorA (63 and 42% identity, respectively), it is located mainly in the periplasm as is TorA; (iii) torZ belongs to the torYZ operon, and the first gene, torY (formerly yecK), encodes a pentahemic c-type cytochrome homologous to the TorC cytochrome of the TorCAD respiratory system. Furthermore, the torYZ operon encodes a third TMAO respiratory system, with catalytic properties that are clearly different from those of the TorCAD and the DmsABC systems. The torYZ and the torCAD operons may have diverged from a common ancestor, but, surprisingly, no torD homologue is found in the sequences around torYZ. Moreover, the torYZ operon is expressed at very low levels under the conditions tested, and, in contrast to torCAD, it is not induced by TMAO or dimethyl sulfoxide.

Reconstitution of the Trimethylamine Oxide Reductase Regulatory Elements of Shewanella oneidensis in Escherichia coli

Several bacteria can grow by using small organic compounds such as trimethylamine oxide (TMAO) as electron acceptors. In Shewanella species, the TMAO reductase respiratory system is encoded by the torECAD operon. We showed that production of the TMAO reductase of S. oneidensis was induced by TMAO and repressed by oxygen, and we noticed that a three-gene cluster (torSTR) encoding a complex two-component regulatory system was present downstream of the torECAD operon. We introduced the torSTR gene cluster into Escherichia coli and showed that this regulatory gene cluster is involved in TMAO induction of the torE promoter but plays no role in the oxygen control. The TorR response regulator was purified, and gel shift and footprinting experiments revealed that TorR binds to a single region located about 70 bases upstream of the transcription start site of the tor structural operon. By deletion analysis, we confirmed that the TorR operator site is required for induction of the tor structural promoter. As the TMAO regulatory system of S. oneidensis is homologous to that of E. coli, we investigated a possible complementation between the TMAO regulatory components of the two bacteria. Interestingly, TorS(ec), the TMAO sensor of E. coli, was able to transphosphorylate TorR(so), the TMAO response regulator of S. oneidensis.

Isolation, organization and expression of the Pseudomonas aeruginosa threonine genes

Three genes from Pseudomonas aeruginosa involved in threonine biosynthesis, hom, thrB and thrC, encoding homoserine dehydrogenase (HDH), homoserine kinase (HK) and threonine synthase (TS), respectively, have been cloned and sequenced. The hom and thrC genes lie at the thr locus of the P. aeruginosa chromosome map (31 min) and are likely to be organized in a bicistronic operon. The encoded proteins are quite similar to the Hom and TS proteins from other bacterial species. The thrB gene was located by pulsed‐field gel electrophoresis experiments at 10 min on the chromosome map. The product of this gene does not share any similarity with other known ThrB proteins. No phenotype could be detected when the chromosomal thrB gene was inactivated by an insertion. Therefore the existence of isozymes for this activity is postulated. HDH activity was feedback inhibited by threonine; the expression of all three genes was constitutive. The overall organization of these three genes appears to differ from that in other bacterial species.

Effets inhibiteurs cooperatifs de la L-lysine avec d’autres amino acides sur une aspartokinase de Escherichia coli

Summary Co-operative inhibitory effects on Escherichia coli lysine-sensitive aspartokinase (ATP: L -aspartate 4-phosphotransferase, EC 2.7.2.4) have been observed. When L -lysine (the „physiological” end-product inhibitor) is employed simultaneously with L -leucine, L -phenylalanine or L -isoleucine, substantial inhibition is obtained. At the concentration used, each, amino acid alone exerts very weak inhibition. The results are discussed in the light of a general theory of allosteric proteins recently put forward.

Cloning in Escherichia coli of genes involved in the synthesis of proline and leucine in Desulfovibrio desulfuricans Norway

SummaryA library of Deusulfovibrio desulfuricans Norway genomic DNA was constructed in Escherichia coli with pBR322 as vector and plasmids able to complement the proA and leuB mutations of the host were screened. It was observed that all the plasmids studied were highly unstable, the insert DNA being rapidely lost under non-selective growth conditions. A 2.75 kb DNA fragment of D. desulfuricans Norway was found to complement E. coli ProA, ProB and ProC deficiencies. From the results of restriction analysis and Southern hybridization, it is proposed that the genes involved in proline and leucine biosynthesis are clustered on the chromosome of D. desulfuricans Norway.

ThrH, a homoserine kinase isozyme with in vivo phosphoserine phosphatase activity in Pseudomonas aeruginosa.

Homoserine kinase, the product of the thrB gene, catalyses an obligatory step of threonine biosynthesis. In Pseudomonas aeruginosa, unlike Escherichia coli, inactivation of the previously identified thrB gene does not result in threonine auxotrophy. A new gene, named thrH, was isolated that, when expressed in E. coli thrB mutant strains, results in complementation of the mutant phenotype. In P. aeruginosa, threonine auxotrophy is observed only when both thrB and thrH are simultaneously inactivated. Thus, thrH encodes a protein with an in vivo homoserine-kinase-like activity. Surprisingly, thrH overexpression allows complementation of serine auxotrophy of E. coli and P. aeruginosa serB mutants. These mutants are affected in the phosphoserine phosphatase protein, an enzyme involved in serine biosynthesis. Comparison analysis revealed sequence homology between ThrH and the SerB proteins from different organisms. This could explain the in vivo phosphoserine phosphatase activity of ThrH when overproduced. ThrH differs from the protein encoded by the serB gene which was identified in P. aeruginosa. Thus, two SerB-like proteins co-exist in P. aeruginosa, a situation also found in Mycobacterium tuberculosis.