Cloning and nucleotide sequences of the homoserine dehydrogenase genes (hom) and the threonine synthase genes (thrC) of the gram-negative obligate methylotroph Methylobacillus glycogenes.

We have cloned the homoserine dehydrogenase genes (hom) from the gram-negative obligate methylotrophs Methylobacillus glycogenes ATCC 21276 and ATCC 21371 by complementation of an Escherichia coli homoserine dehydrogenase-deficient mutant. The 4.15-kb DNA fragment cloned from M. glycogenes ATCC 21371 also complemented an E. coli threonine synthase-deficient mutant, suggesting the DNA fragment contained the thrC gene in addition to the hom gene. The homoserine dehydrogenases expressed in the E. coli recombinants were hardly inhibited by L-threonine, L-phenylalanine, or L-methionine. However, they became sensitive to the amino acids after storage at 4 degrees C for 4 days as in M. glycogenes. The structures of the homoserine dehydrogenases overexpressed in E. coli were thought to be different from those in M. glycogenes, probably in subunit numbers of the enzyme, and were thought to have converted to the correct structures during the storage. The nucleotide sequences of the hom and thrC genes were determined. The hom genes of M. glycogenes ATCC 21276 and ATCC 21371 encode peptides with M(r)s of 48,225 and 44,815, respectively. The thrC genes were located 50 bp downstream of the hom genes. The thrC gene of ATCC 21371 encodes a peptide with an M(r) of 52,111, and the gene product of ATCC 21276 was truncated. Northern (RNA) blot analysis suggests that the hom and thrC genes are organized in an operon. Significant homology between the predicted amino acid sequences of the hom and thrC genes and those from other microorganisms was found.     

Evolution of biosynthetic pathways: a common ancestor for threonine synthase, threonine dehydratase and D-serine dehydratase.

The Bacillus subtilis genes encoding threonine synthase (thrC) and homoserine kinase (thrB) have been cloned via complementation of Escherichia coli thr mutants. Determination of their nucleotide sequences indicates that the thrC stop codon overlaps the thrB start codon; this genetic organization suggests that the two genes belong to the same operon, as in E. coli. However, the gene order is thrC-thrB in B. subtilis whereas it is thrB-thrC in the thr operon of E. coli. This inversion of the thrC and thrB genes between E. coli and B. subtilis is indicative of a possible independent construction of the thr operon in these two organisms. In other respects, comparison of the predicted amino acid sequences of the B. subtilis and E. coli threonine synthases with that of Saccharomyces cerevisiae threonine dehydratase and that of E. coli D-serine dehydratase revealed extensive homologies between these pyridoxal phosphate-dependent enzymes. This sequence homology, which correlates with similarities in the catalytic mechanisms of these enzymes, indicates that these proteins, catalyzing different reactions in different metabolic pathways, may have evolved from a common ancestor.     

Detection of the homology among proteins by immunochemical cross-reactivity between denatured antigens. Application to the threonine and methionine regulated aspartokinases-homoserine dehydrogenases from Escherichia coli K 12.

The two isofunctional enzymes aspartokinases-homoserine dehydrogenases I and II from Escherichia coli K 12 are compared using immunochemical techniques. The antibodies raised against one of these two proteins when in its native state can only recognize the homologous antigen, whether it is native or denatured. Contrarily, the antibodies raised against one of these two proteins when in its denatured state can recognize both the homologous and heterologous denatured antigens. The existence of this cross-reaction only between the two denatured aspartokinases-homoserine dehydrogenases suggests that these two enzymes have some similarity since such a reaction is not detected with several other denatured proteins. The regions involved in this similarity are buried inside the native proteins, and become exposed only upon denaturation. The same results, the existence of a cross-reaction between denatured species and none between the native ones, is obtained with proteolytic fragments derived from these two proteins and endowed with homoserine dehydrogenase activity. This resemblance between the two aspartokinases-homoserine dehydrogenases suggests that these proteins derive from a common ancestor. It is also proposed that such a cross-reaction between two denatured proteins is evidence for an homology between their amino acid sequences, and that the use of denatured proteins as both immunogens and antigens could be useful in detecting sequence homologies.     

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.     

Immunological cross reactivity of four enzymes involved in the biosynthetic pathway of lysine, methionine and threonine in Escherichia coli K12.

In Escherichia coli K12 the biosynthetic pathway of lysine, methionine and threonine is characterized by three isofunctional aspartokinases and two homoserine dehydrogenases. A single polypeptide chain carries the threonine-sensitive aspartokinase and homoserine dehydrogenase (AK I-HDH I), and a different polypeptide chain carries the methionine-repressible aspartokinase and homoserine dehydrogenase (AK II-HDH II). Immuno-adsorbants prepared with rabbit antibodies against AK I-HDH I bind the lysine-sensitive aspartokinase (AK III), the AK II-HDH II, and the homoserine kinase (HSK), an enzyme of the threonine biosynthetic pathway. Saturation of the immunoadsorbant with AK I-HDH I results in a decreased binding capacity for the other enzymes. Displacement of bound AK III or HSK can be obtained with pure AK I-HDH I, showing that the affinity of the antibodies to homologous antigens is higher than to heterologous ones. Immunoadsorbants prepared with anti-HSK antibodies show the same type of recognition: binding of the three aspartkinases and a capacity to displace the heterologous antigens bound. Accordingly, the same antibodies, implicated in the binding of the homologous antigen, bind the other enzymes. None of the other enzymes of the pathway, or the other kinases tested are recognized by the two immunoadsorbants. It can be postulated that in E. coli K12, duplication of a common ancestor gene gave rise to the three aspartokinases and to the homoserine kinase; two of the genes coding for the aspartokinases fused with those coding for the homoserine dehydrogenases. Indicating that only few epitopes are shared by these enzymes, by conventional immuno-diffusion techniques no precipitation lines appeared with antibodies against AK I-HDH I and the other proteins.     

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.     

Nucleotide sequence of the thrB gene of E. coli, and its two adjacent regions; the thrAB and thrBC junctions.

We have sequenced a DNA fragment containing the Escherichia coli thrA-thrB junction, the complete thrB gene and the thrB-thrC junction. The intergenic sequence thrA and thrB is only one base pair. The coding region for homoserine kinase is 927 base pairs long. It is followed by 114 base pair segment in an open reading frame predicting that thrC begins just after non-sense codon of thrB. The presence at the end of thrA and of thrB of sequences that can pair with the 3' end of the 16 S ribosomal RNA suggests that reinitiation of translation occurs at the end of the two genes. The deduced aminoacid sequence for homoserine kinase shows no striking homology with aspartokinase I homoserine dehydrogenase I.     

A C-terminal deletion in Corynebacterium glutamicum homoserine dehydrogenase abolishes allosteric inhibition by L-threonine.

In Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum, homoserine dehydrogenase (HD), the enzyme after the branch point of the threonine/methionine and lysine biosynthetic pathways, is allosterically inhibited by L-threonine. To investigate the regulation of the C. glutamicum HD enzyme by L-threonine, the structural gene, hom, was mutated by UV irradiation of whole cells to obtain a deregulated allele, homdr. L-Threonine inhibits the wild-type (wt) enzyme with a Ki of 0.16 mM. The deregulated enzyme remains 80% active in the presence of 50 mM L-threonine. The homdr gene mutant was isolated and cloned in E. coli. In a C. glutamicum wt host background, but not in E. coli, the cloned homdr gene is genetically unstable. The cloned homdr gene is overexpressed tenfold in C. glutamicum and is active in the presence of over 60 mM L-threonine. Sequence analysis revealed that the homdr mutation is a single nucleotide (G1964) deletion in codon 429 within the hom reading frame. The resulting frame-shift mutation radically alters the structure of the C terminus, resulting in ten amino acid (aa) changes and a deletion of the last 7 aa relative to the wt protein. These observations suggest that the C terminus may be associated with the L-threonine allosteric response. The homdr mutation is unstable and probably deleterious to the cell. This may explain why only one mutation was obtained despite repeated mutagenesis.     

Mapping of the structural genes of the three aspartokinases and of the two homoserine dehydrogenases of Escherichia coli K-12

Mutants requiring threonine plus methionine (or homoserine), or threonine plus methionine plus diaminopimelate (or homoserine plus diaminopimelate) have been isolated from strains possessing only one of the three isofunctional aspartokinases. They have been classified in several groups according to their enzymatic defects. Their mapping is described. Several regions of the chromosome are concerned: thrA (aspartokinase I-homoserine dehydrogenase I) is mapped in the same region as thrB and thrC (0 min). lysC (aspartokinase III) is mapped at 80 min, far from the other genes coding for diaminopimelate synthesis. metLM (aspartokinase II-homoserine dehydrogenase II) lies at 78 min closely linked to metB, metJ, and metF.     

Cloning, expression, and sequence analysis of the Bacillus methanolicus C1 methanol dehydrogenase gene.

The gene (mdh) coding for methanol dehydrogenase (MDH) of thermotolerant, methylotroph Bacillus methanolicus C1 has been cloned and sequenced. The deduced amino acid sequence of the mdh gene exhibited similarity to those of five other alcohol dehydrogenase (type III) enzymes, which are distinct from the long-chain zinc-containing (type I) or short-chain zinc-lacking (type II) enzymes. Highly efficient expression of the mdh gene in Escherichia coli was probably driven from its own promoter sequence. After purification of MDH from E. coli, the kinetic and biochemical properties of the enzyme were investigated. The physiological effect of MDH synthesis in E. coli and the role of conserved sequence patterns in type III alcohol dehydrogenases have been analyzed and are discussed.     

Transductional construction of a threonine-hyperproducing strain of Serratia marcescens: lack of feedback controls of three aspartokinases and two homoserine dehydrogenases.

To construct a threonine-hyperproducing strain of Serratia marcescens Sr41, the six regulatory mutations for three aspartokinases and two homoserine dehydrogenases were combined in a single strain by three transductional crosses. The constructed strain, T-1026, carried the lysC1 mutation leading to lack of feedback inhibition and repression of aspartokinase III, the thrA1(1) mutation desensitizing aspartokinase I to feedback inhibition, the thrA2(1) mutation releasing feedback inhibition of homoserine dehydrogenase I, the two hnr mutations derepressing aspartokinase I and homoserine dehydrogenase I, and the etr-1 mutation derepressing aspartokinase II and homoserine dehydrogenase II. The strain produced ca. 40 mg of threonine per ml of medium containing sucrose and urea. Furthermore, the productivity of strain T-1026 was compared with those of strains devoid of more than one of the six regulatory mutations.     

Transductional construction of a threonine-hyperproducing strain of Serratia marcescens: lack of feedback controls of three aspartokinases and two homoserine dehydrogenases

To construct a threonine-hyperproducing strain of Serratia marcescens Sr41, the six regulatory mutations for three aspartokinases and two homoserine dehydrogenases were combined in a single strain by three transductional crosses. The constructed strain, T-1026, carried the lysC1 mutation leading to lack of feedback inhibition and repression of aspartokinase III, the thrA1(1) mutation desensitizing aspartokinase I to feedback inhibition, the thrA2(1) mutation releasing feedback inhibition of homoserine dehydrogenase I, the two hnr mutations derepressing aspartokinase I and homoserine dehydrogenase I, and the etr-1 mutation derepressing aspartokinase II and homoserine dehydrogenase II. The strain produced ca. 40 mg of threonine per ml of medium containing sucrose and urea. Furthermore, the productivity of strain T-1026 was compared with those of strains devoid of more than one of the six regulatory mutations.     

Interaction of substrates and inhibitors with the homoserine dehydrogenase of kinase-inactivated aspartokinase I.

The aspartokinase activity of the aspartokinase-homoserine dehydrogenase complex of Escherichia coli was affinity labeled with substrates ATP, aspartate, and feedback inhibitor threonine. Exchange-inert ternary adducts of Co(III)-aspartokinase and either ATP, aspartate or threonine were formed by oxidation of corresponding Co(II) ternary complexes with H2O2. The ternary enzyme-Co(III)-threonine adduct (I) had 3.8 threonine binding sites per tetramer, one-half that of the native enzyme. The binding of threonine to I was still cooperative as determined by equilibrium dialysis (nH = 2.2) or by studying inhibition of residual dehydrogenase activity (nH = 2.7). Threonine still protected the SH groups of I against 5,5'-dithiobis(2-nitrobenzoate) (DTNB) reaction but the number of SH groups reacting with thiol reagents (DTNB) was reduced by 1-2 per subunit in the absence of threonine. This suggests either that Co(III) is bound to the enzyme via sulfhydryl groups or that 1-2SH groups are buried or rendered inaccessible in I. The binding of threonine to sites not blocked by the affinity labeling produced changes in the circular dichroism of the complex comparable to changes produced by threonine binding to native enzyme and also protected against proteolytic digestion. The major conformational changes produced by threonine are thus ascribable to binding at this one class of regulatory sites. The interactions of kinase substrates with various aspartokinase-Co(III) complexes containing ATP, aspartate, or threonine and a threonine-insensitive homoserine dehydrogenase produced by mild proteolysis were studied. The inhibition of homoserine dehydrogenase by kinase substrates is not due to binding of these inhibitors at the kinase active site but was shown to be due to binding to sites within the dehydrogenase domain of the enzyme. L-alpha-Aminobutyrate, a presumed threonine analogue, also inhibits the dehydrogenase by binding at the same or similar sites in the dehydrogenase domain and not at threonine regulatory site.     

Cloning, expression, and isolation of the mannitol transport protein from the thermophilic bacterium Bacillus stearothermophilus.

A mannitol phosphotransferase system (PTS) was identified in Bacillus stearothermophilus by in vitro complementation with Escherichia coli EI, HPr, and IIA(Mtl). Degenerate primers based on regions of high amino acid similarity in the E. coli and Staphylococcus carnosus EII(Mt1) were used to develop a digoxigenin-labeled probe by PCR. Using this probe, we isolated three overlapping DNA fragments totaling 7.2 kb which contain the genes mtlA, mtlR, mtlF, and mtlD, encoding the mannitol IICB,a regulator, IIA, and a mannitol-1-phosphate dehydrogenase, respectively. The mtl4 gene consists of 1,413 bp coding for a 471-amino-acid protein with a calculated mass of 50.1 kDa. The amino acid sequence shows high similarity with the sequence of IICB(Mtl) of S. carnosus and the IICB part of the IICBA(Mtl)s of E. coli and B. subtilis. The enzyme could be functionally expressed in E. coli by placing it behind the strong tac promoter. The rate of thermal inactivation at 60 degrees C of B. stearothermophilus HCB(Mt1) expressed in E. coli was two times lower than that of E. coli IICB(Mtl). IICB(Mtl) in B. stearothermophilus is maximally active at 85 degrees C and thus very thermostable. The enzyme was purified on Ni-nitrilotriacetic acid resin to greater than 95% purity after six histidines were fused to the C-terminal part of the transporter.     

Genetic characterization of Bacillus subtilis odhA and odhB, encoding 2-oxoglutarate dehydrogenase and dihydrolipoamide transsuccinylase, respectively.

The 2-oxoglutarate dehydrogenase complex consists of three different subenzymes, the E1o (2-oxoglutarate dehydrogenase) component, the E2o (dihydrolipoyl transsuccinylase) component, and the E3 (dihydrolipoamide dehydrogenase) component. In Bacillus subtilis, the E1o and E2o subenzymes are encoded by odhA and odhB, respectively. A plasmid with a 6.8-kilobase-pair DNA fragment containing odhA and odhB was isolated. Functional E1o and E2o are expressed from this plasmid in Escherichia coli. Antisera generated against B. subtilis E1o and E2o expressed in E. coli reacted with antigens of the same size from B. subtilis. The nucleotide sequence of odhB and the terminal part of odhA was determined. The deduced primary sequence of B. subtilis E2o shows striking similarity to the corresponding E. coli protein, which made it possible to identify the lipoyl-binding lysine residue as well as catalytic histidine and aspartic acid residues. An mRNA of 4.5 kilobases hybridizing to both odhA and odhB probes was detected, indicating that odhA and odhB form an operon.     

Lipoic acid synthesis: a new family of octanoyltransferases generally annotated as lipoate protein ligases

Bacillus subtilis lacks a recognizable homologue of the LipB octanoyltransferase, an enzyme essential for lipoic acid synthesis in Escherichia coli. LipB transfers the octanoyl moiety from octanoyl-acyl carrier protein to the lipoyl domains of the 2-oxoacid dehydrogenases via a thioester-linked octanoyl-LipB intermediate. The octanoylated dehydrogenase is then converted to the enzymatically active lipoylated species by insertion of two sulfur atoms into the octanoyl moiety by the S-adenosyl-l-methionine radical enzyme, LipA (lipoate synthase). B. subtilis synthesizes lipoic acid and contains a LipA homologue that is fully functional in E. coli. Therefore, the lack of a LipB homologue presented the puzzle of how B. subtilis synthesizes the LipA substrate. We report that B. subtilis encodes an octanoyltransferase that has virtually no sequence resemblance to E. coli LipB but instead has a sequence that resembles that of the E. coli lipoate ligase, LplA. On the basis of this resemblance, these genes have generally been annotated as encoding a lipoate ligase, an enzyme that in E. coli scavenges lipoic acid from the environment but plays no role in de novo synthesis. We have named the B. subtilis octanoyltransferase LipM and find that, like LipB, the LipM reaction proceeds through a thioester-linked acyl enzyme intermediate. The LipM active site nucleophile was identified as C150 by the finding that this thiol becomes modified when LipM is expressed in E. coli. The level of the octanoyl-LipM intermediate can be significantly decreased by blocking fatty acid synthesis during LipM expression, and C150 was confirmed as an essential active site residue by site-directed mutagenesis. LipM homologues seem the sole type of octanoyltransferase present in the firmicutes and are also present in the cyanobacteria. LipM type octanoyltransferases represent a new clade of the PF03099 protein family, suggesting that octanoyl transfer activity has evolved at least twice within this superfamily.     

Mannitol-specific phosphoenolpyruvate-dependent phosphotransferase system of Enterococcus faecalis: molecular cloning and nucleotide sequences of the enzyme IIIMtl gene and the mannitol-1-phosphate dehydrogenase gene, expression in Escherichia coli, and comparison of the gene products with similar enzymes.

Enzyme IIIMtl is part of the mannitol phosphotransferase system of Enterococcus faecalis. It is phosphorylated in a reaction sequence requiring enzyme I and heat-stable phosphocarrier protein (HPr). The phospho group is transferred from enzyme IIIMtl to enzyme IIMtl, which then catalyzes the uptake and concomitant phosphorylation of mannitol. The internalized mannitol-1-phosphate is oxidized to fructose-6-phosphate by mannitol-1-phosphate dehydrogenase. In this report we describe the cloning of the mtlF and mtlD genes, encoding enzyme IIIMtl and mannitol-1-phosphate dehydrogenase of E. faecalis, by a complementation system designed for cloning of gram-positive phosphotransferase system genes. The complete nucleotide sequences of mtlF, mtlD, and flanking regions were determined. From the gene sequences, the primary translation products are deduced to consist of 145 amino acids (enzyme IIIMtl) and 374 amino acids (mannitol-1-phosphate dehydrogenase). Amino acid sequence comparison confirmed a 41% similarity of E. faecalis enzyme IIIMtl to the hydrophilic enzyme IIIMtl-like portion of enzyme IIMtl of Escherichia coli and 45% similarity to enzyme IIIMtl of Staphylococcus carnosus. The putative N-terminal NAD+ binding domain of mannitol-1-phosphate dehydrogenase of E. faecalis shows a high degree of similarity with the N terminus of E. coli mannitol-1-phosphate dehydrogenase (T. Davis, M. Yamada, M. Elgort, and M. H. Saier, Jr., Mol. Microbiol. 2:405-412, 1988) and the N-terminal part of the translation product of S. carnosus mtlD, which was also determined in this study. There is 40% similarity between the dehydrogenases of E. faecalis and E. coli over the whole length of the enzymes. The organization of mannitol-specific genes in E. faecalis seems to be similar to the organization in S. carnosus. The open reading frame for enzyme IIIMtl E. faecalis is followed by a stem-loop structure, analogous to a typical Rho-independent terminator. We conclude that the mannitol-specific genes are organized in an operon and that the gene order is mtlA orfX mtlF mtlD.