Crc (catabolite repression control) protein of Pseudomonas aeruginosa has shown to be involved in carbon regulation of several pathways. In this study, the role of Crc in catabolite repression control has been studied in Pseudomonas putida. The bkd operons of P. putida and P. aeruginosa encode the inducible multienzyme complex branched-chain keto acid dehydrogenase, which is regulated in both species by catabolite repression. We report here that this effect is mediated in both species by Crc. A 13-kb cloned DNA fragment containing the P. putida crc gene region was sequenced. Crc regulates the expression of branched-chain keto acid dehydrogenase, glucose-6-phosphate dehydrogenase, and amidase in both species but not urocanase, although the carbon sources responsible for catabolite repression in the two species differ. Transposon mutants affected in their expression of BkdR, the transcriptional activator of the bkd operon, were isolated and identified as crc and vacB (rnr) mutants. These mutants suggested that catabolite repression in pseudomonads might, in part, involve control of BkdR levels.Pseudomonads play an important role in nature because of their ability to metabolize natural and manufactured organic chemicals. Many of these compounds are environmental pollutants, such as benzene, toluene, xylene, ethylbenzene, styrene, and chlorobenzoates (18), and their removal has been named bioremediation. Although the enzymic pathways responsible for degradation of these pollutants may be effective when the target compound is the sole growth-supporting substrate, in nature these compounds are present as mixtures, and some substrates may be degraded preferentially. Catabolite repression control refers to the ability of an organism to preferentially metabolize one carbon source over another when both are present in the growth medium. Because of the importance of pseudomonads to bioremediation efforts, understanding the control of catabolite repression is important so that more efficient, genetically modified organisms can be utilized in the removal of these environmental pollutants.The molecular mechanisms of catabolite repression control have been extensively characterized in enteric bacteria, where glucose is the preferred carbon source. In these organisms, enzymes of the phosphoenolpyruvate-dependent phosphotransferase system mediate catabolite repression control by regulation of cyclic AMP (cAMP) concentration via adenylate cyclase activity (22). The strongest repressing substrates in Pseudomonas spp. are acetate, tricarboxylic acid cycle intermediates, and glucose (4, 10, 26). Unlike Escherichia coli, in Pseudomonas species adenylate cyclase activity, cAMP phosphodiesterase activity, and cAMP pools do not fluctuate with carbon source, nor does the addition of cAMP relieve repression of catabolite responsive pathways (21, 25). In addition, only one phosphotransferase system (fructose) has been identified in Pseudomonas (5), suggesting that PTS components are not involved in catabolite repression control in pseudomo...

The three-dimensional structure of one of the three lipoamide dehydrogenases occurring in Pseudomonas putida, LipDH Val, has been determined at 2.45 A resolution. The orthorhombic crystals, grown in the presence of 20 mM NAD+, contain 458 residues per asymmetric unit. A crystallographic 2-fold axis generates the dimer which is observed in solution. The final crystallographic R-factor is 21.8% for 18,216 unique reflections and a model consisting of 3,452 protein atoms, 189 solvent molecules and 44 NAD+ atoms, while the overall B-factor is unusually high: 47 A2. The structure of LipDH Val reveals the conformation of the C-terminal residues which fold "back" into the putative lipoamide binding region. The C-terminus has been proven to be important for activity by site-directed mutagenesis. However, the distance of the C-terminus to the catalytically essential residues is surprisingly large, over 6 A, and the precise role of the C-terminus still needs to be elucidated. In this crystal form LipDH Val contains one NAD+ molecule per subunit. Its adenine-ribose moiety occupies an analogous position as in the structure of glutathione reductase. However, the nicotinamide-ribose moiety is far removed from its expected position near the isoalloxazine ring and points into solution. Comparison of LipDH Val with Azotobacter vinelandii lipoamide dehydrogenase yields an rms difference of 1.6 A for 440 well defined C alpha atoms per subunit. Comparing LipDH Val with glutathione reductase shows large differences in the tertiary and quaternary structure of the two enzymes. For instance, the two subunits in the dimer are shifted by 6 A with respect to each other. So, LipDH Val confirms the surprising differences in molecular architecture between glutathione reductase and lipoamide dehydrogenase, which were already observed in Azotobacter vinelandii LipDH. This is the more remarkable since the active sites are located at the subunit interface and are virtually identical in all three enzymes.

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The effect of growth in 2xYT medium on catabolite repression control in Pseudomonas putida has been investigated using the bkd operon, encoding branched-chain keto acid dehydrogenase. Crc (catabolite repression control protein) was shown to be responsible for repression of bkd operon transcription in 2xYT. BkdR levels were elevated in a P. putida crc mutant, but bkdR transcript levels were the same in both wild type and crc mutant. This suggests that the mechanism of catabolite repression control in rich media by Crc involves posttranscriptional regulation of the bkdR message.The molecular mechanisms of catabolite repression have been well described in enteric bacteria, where enzymes of the phosphoenolpyruvate phosphotransferase system mediate catabolite repression control by regulation of cAMP concentration via adenylate cyclase activity (19). However, a similar mechanism does not appear to be present in Pseudomonas because adenylate cyclase activity and cAMP pools do not fluctuate with carbon source, nor does addition of cAMP relieve repression of catabolite responsive pathways (12, 18). The only protein thus far shown to be involved in catabolite repression in Pseudomonas is Crc of P. aeruginosa, but a function has not been identified (13). However, Crc does not appear to bind DNA (13), suggesting that it is not simply a DNA-binding negative regulator.Crc is involved in catabolite repression of P. putida branched-chain keto acid dehydrogenase (BCKAD), glucose-5-phosphate dehydrogenase, and amidase by glucose and succinate in synthetic media (11). BCKAD is encoded by the four structural genes of the bkd operon, which is positively regulated by BkdR (15). BkdR is a homologue of Lrp (leucineresponsive protein), which is a global transcriptional regulator in Escherichia coli (4). However, pseudomonads and enteric bacteria live in complex media in nature and not in chemically defined media. Expression of lrp is downregulated in nutritionally rich media (6), which suggested that this might also be the case with bkdR. In this report, the effect of 2xYT medium on the expression of bkdR in wild type and in a crc mutant of P. putida was studied to determine if catabolite repression control of the bkd operon might be accomplished by controlling the level of BkdR in the cell.Crc downregulates BCKAD activity in 2xYT. The wild-type strains of P. putida and P. aeruginosa, their crc mutants, and the complemented mutants (11) were grown to an A 660 of ϳ0.6 in 100 ml of 2xYT plus 0.3% valine and 0.1% isoleucine (wt/vol) and then harvested; cell extracts were then prepared as described earlier (16). P. putida JS394 had five-to sixfold higher activity than either PpG2 or JS394 (pJRS196) ( Table 1), and a similar result was obtained when BCKAD activity of PAO8020 was compared to the activities of PAO1 and PAO8020 (pPZ352). These results demonstrate that Crc is involved in catabolite repression control of BCKAD activity by 2xYT in both P. putida and P. aeruginosa. However, the BCKAD activities of the crc were much lower than that ...

Branched-chain keto acid dehydrogenase is a multienzyme complex which is required for the metabolism of the branched-chain amino acids in Pseudomonas putida. The Branched-chain keto acid dehydrogenase is an enzyme which is common to the metabolism of valine, leucine, and isoleucine in Pseudomonas putida (7) and eukaryotes (10,30). It catalyzes the oxidative decarboxylation of branchedchain keto acids formed by transamination of branchedchain amino acids. The reaction with 2-ketoisovalerate, formed from valine, is 2-ketoisovalerate + NAD+ + CoASH -> isobutyryl coenzyme A + CO2 + NADH + H'. The enzyme has been characterized from several sources, including P. putida (36), P. aeruginosa (24), bovine kidney (31), rabbit liver (29), rat kidney (27), and Bacillus subtilis (19). In the latter organism, it functions as a combined pyruvate and branched-chain keto acid dehydrogenase. Branched-chain keto acid dehydrogenase is a multienzyme complex which is composed of four proteins, Elao, E1l3, E2, and E3. The Elac4 component is the dehydrogenase and decarboxylase, the E2 component catalyzes transacylation between its lipoyl residue and coenzyme A, and the E3 component is lipoamide dehydrogenase, which catalyzes the oxidation of the lipoyl residue of E2. The structure of keto acid dehydrogenase complexes has been reviewed elsewhere (10,23,30

We cloped the structural genes for the individual subunits of the branched-chain keto acid dehydrogenase multienzyme complex on a 7.8-kilobase EcoRI-SstI restriction fragment of Pseudomonas putida chromosomal DNA by cloning into the broad-host-range vector pKT230. A direct selection system for growth on valine-isoleucine agar was achieved by complementation of P. putida branched-chain keto acid dehydrogenawe mutants. The recombinant plasmid, pSS1-1, increased expression of branched-chain ketQ acid dehydrogenase up to five times in wild-type P. putida. The complex was expressed cQnstitutively in P. putida(pSSl-l) but was inducible in Escherichia coli HB101(pSS1-1) by high valine. E. coli minicells tra'nsformed with pSS1-1 produced three polypeptides which did not match the fopr polypeptides of the purified complex. To resolve this problem, we inserted P. putida DNA from pSS1-i into pUC18 And pUC19. The pUC-derived plasmids were used as DNA templates in an E. coli transcription-translation system. Four polypeptides were produced from the pUC18-derived plasmid which had the correct molecular weights, showing that the structural genes had been cloned. Since only weak bands were produced with the pUC19-derived plasmid, the direction of transcription was established. The locations and order of all the structural genes of branched-chain keto acid dehydrogenase were located by restriction enzyme mapping.Branched-chain keto acid dehydrogenase is an enzyme common to the catabolism of valine, leucine, and isoleucine. The enzyme has been purified from mammals (25, 26) and from Pseudomonas putida (31), Pseudomonas aeruginosa (21), and Bacillus subtilis (17). In each species, it is a multienzyme complex composed of three functional subunits: El, the dehydrogenase-decarboxylase; E2, the transacylase; and E3, lipoamide dehydrogenase. Purified branched-chain keto acid dehydrogena'se from mammnals is composed of four polypeptides, the E-1 subunit being composed of two dissimilar proteins. The E2 and E3 subunits of the Pseudomonas complexes have been identified, but it was not clear whether El consisted of one or two polypeptides. P. putida and P. aeruginosa are unusual in that they possess two functionally and structurally distinct lipoamide dehydrogenases, 29). LPD-Val is the specific E3 subunit of branched-chain keto acid dehydrogenase. Mutations affecting subunits of branched-chain keto acid dehydrogenase including LPD-Val map by conjugation at a single location on the P. putida chromosomes, suggesting that the structural genes are linked (37). LPD-Glc is the E3 subunit of 2-ketoglutarate and probably pyruvate dehydrogenase and is the L-factor of glycine decarboxylase in P.putida (28,29). In Escherichia coli there is a single lipoamide dehydrogenase which functions as the E3 subunit of the pyruvate and 2-ketoglutarate dehydrogrnases (11).There is some evidence which suggests that branchedchain keto acid dehydrogenase evolved from pyruvate dehydrogenase. Lowe et al. (17) have isolated a dual-purpose keto acid dehydrogenase fro...

The bkd operon of Pseudomonas putida consists of the structural genes encoding the components of the inducible branched-chain ketoacid dehydrogenase. BkdR, a positive regulator of the bkd operon and a homolog of Lrp of Escherichia coli is encoded by a structural gene adjacent to, and divergently transcribed from, the bkd operon of P. putida. BkdR was purified from E. coli containing bkdR cloned into pCYTEXP1, an expression vector. The molecular weight of BkdR obtained by gel filtration indicates that BkdR is a tetramer, and the abundance of BkdR in P. putida was estimated to be about 25 to 40 copies of the tetramer per cell. BkdR bound specifically to the region between bkdR and bkdA1, the latter being the first gene of the bkd operon. One BkdR-DNA complex was observed in gel mobility shift patterns. Approximately 100 bp was protected from the action of DNase I by BkdR, and the addition of L-branched-chain amino acids enhanced the appearance of hypersensitive sites in the protected region. There are four potential BkdR-DNA binding sequences in this region based on similarity to Lrp-binding consensus sequences. Like many other transcriptional activators, BkdR regulates expression of its structural gene. DNAs from several gram-negative bacteria hybridized to a probe containing bkdR, indicating the presence of bkdR-like genes in these organisms.Branched-chain ketoacid dehydrogenase of Pseudomonas putida is an inducible multienzyme complex produced by the organism grown in media with branched-chain amino or keto acids as carbon sources (15). The formation of branched-chain ketoacid dehydrogenase is repressible by glucose and NH 4 ϩ (27), and the effects seem to be additive. The components of branched-chain ketoacid dehydrogenase are E1 (the dehydrogenase-decarboxylase), E2 (the transacylase), and E3 (lipoamide dehydrogenase). The structural genes for these proteins are encoded by the bkd operon of P. putida, which has been cloned and whose nucleotide sequence has been determined (3-5). All four genes are tightly linked, and the operon is transcribed as a polycistronic message (5).An open reading frame upstream of, and divergently transcribed from, the bkd operon, encoding a protein, BkdR (13), with 37.5% amino acid sequence identity to Lrp, the leucineresponsive regulatory protein of Escherichia coli (33), was found. Lrp is a global transcriptional regulator in E. coli (6), the action of which can be antagonized or potentiated by leucine or may be insensitive to the presence of leucine. Expression of the ilvIH operon of E. coli is regulated by Lrp and is repressible by leucine. Lrp binds to several sites in the regulatory region of the ilvIH operon of E. coli (29) and causes DNA bending (30).Chromosomal mutations affecting bkdR resulted in failure to produce branched-chain ketoacid dehydrogenase, and mutations in bkdR were complemented in trans by both bkdR and lrp (13). These results suggest that BkdR acts as a positive transcriptional activator of the bkd operon. This article describes the cloning and overexpressi...

The activities of six enzymes which take part in the oxidation of valine by Pseudomonas putida were measured under various conditions of growth. The formation of four of the six enzymes was induced by growth on d - or l -valine: d -amino acid dehydrogenase, branched-chain keto acid dehydrogenase, 3-hydroxyisobutyrate dehydrogenase, and methylmalonate semialdehyde dehydrogenase. Branched-chain amino acid transaminase and isobutyryl-CoA dehydrogenase were synthesized constitutively. d -Amino acid dehydrogenase and branched-chain keto acid dehydrogenase were induced during growth on valine, leucine, and isoleucine, and these enzymes were assumed to be common to the metabolism of all three branched-chain amino acids. The segment of the pathway required for oxidation of isobutyrate was induced by growth on isobutyrate or 3-hydroxyisobutyrate without formation of the preceding enzymes. d -Amino acid dehydrogenase was induced by growth on l -alanine without formation of other enzymes required for the catabolism of valine. d -Valine was a more effective inducer of d -amino acid dehydrogenase than was l -valine. Therefore, the valine catabolic pathway was induced in three separate segments: (i) d -amino acid dehydrogenase, (ii) branched-chain keto acid dehydrogenase, and (iii) 3-hydroxyisobutyrate dehydrogenase plus methylmalonate semialdehyde dehydrogenase. In a study of the kinetics of formation of the inducible enzymes, it was found that 3-hydroxyisobutyrate and methylmalonate semialdehyde dehydrogenases were coordinately induced. Induction of enzymes of the valine catabolic pathway was studied in a mutant that had lost the ability to grow on all three branched-chain amino acids. Strain PpM2106 had lowered levels of branched-chain amino acid transaminase and completely lacked branched-chain keto acid dehydrogenase when grown in medium which contained valine. Addition of 2-ketoisovalerate, 2-ketoisocaproate, or 2-keto-3-methylvalerate to the growth medium of strain PpM2106 resulted in induction of normal levels of branched-chain keto acid dehydrogenase; therefore, the branched-chain keto acids were the actual inducers of branched-chain keto acid dehydrogenase.