The djlA gene acts synergistically with dnaJ in promoting Escherichia coli growth.

The DnaK chaperone of Escherichia coli is known to interact with the J domains of DnaJ, CbpA, and DjlA. By constructing multiple mutants, we found that the djlA gene was essential for bacterial growth above 37 degrees C in the absence of dnaJ. This essentiality depended upon the J domain of DjlA but not upon the normal membrane location of DjlA.

The importance of a mobile loop in regulating chaperonin/ co-chaperonin interaction: humans versus Escherichia coli.

Chaperonins are universally conserved proteins that nonspecifically facilitate the folding of a wide spectrum of proteins. While bacterial GroEL is functionally promiscuous with various co-chaperonin partners, its human homologue, Hsp60 functions specifically with its co-chaperonin partner, Hsp10, and not with other co-chaperonins, such as the bacterial GroES or bacteriophage T4-encoded Gp31. Co-chaperonin interaction with chaperonin is mediated by the co-chaperonin mobile loop that folds into a beta-hairpin conformation upon binding to the chaperonin. A delicate balance of flexibility and conformational preferences of the mobile loop determines co-chaperonin affinity for chaperonin. Here, we show that the ability of Hsp10, but not GroES, to interact specifically with Hsp60 lies within the mobile loop sequence. Using mutational analysis, we show that three substitutions in the GroES mobile loop are necessary and sufficient to acquire Hsp10-like specificity. Two of these substitutions are predicted to preorganize the beta-hairpin turn and one to increase the hydrophobicity of the GroEL-binding site. Together, they result in a GroES that binds chaperonins with higher affinity. It seems likely that the single ring mitochondrial Hsp60 exhibits intrinsically lower affinity for the co-chaperonin that can be compensated for by a higher affinity mobile loop.

Identification and characterization of HsIV HsIU (ClpQ ClpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli.

Heat shock response in Escherichia coli is autoregulated. Consistent with this, mutations in certain heat shock genes, such as dnaK, dnaJ, grpE or htrC lead to a higher constitutive heat shock gene expression at low temperatures. A similar situation occurs upon accumulation of newly synthesized peptides released prematurely from the ribosomes by puromycin. We looked for gene(s) which, when present in multicopy, prevent the constitutive heat shock response associated with htrC mutant bacteria or caused by the presence of puromycin. One such locus was identified and shown to carry the recently sequenced hslV hslU (clpQ clpY) operon. HslV/ClpQ shares a very high degree of homology with members of the beta-type subunit, constituting the catalytic core of the 20S proteasome. HslU/ClpY is 50% identical to the ClpX protein of E. coli, which is known to present large polypeptides to its partner, the ATP-independent proteolytic enzyme ClpP. We show that, in vivo, HslV and HslU interact and participate in the degradation of abnormal puromycylpolypeptides. Biochemical evidence suggests that HslV/ClpQ is an efficient peptidase whose activity is enhanced by HslU/CIpY in the presence of ATP.

Identification and characterization of a new disulfide isomerase-like protein (DsbD) in Escherichia coli.

Previous studies have established that DsbA and DsbC, periplasmic proteins of Escherichia coli, are two key players involved in disulfide bond formation. A search for extragenic mutations able to compensate for the lack of dsbA function in vivo led us to the identification of a new gene, designated dsbD. Lack of DsbD protein leads to some, but not all, of the phenotypic defects observed with other dsb mutations, such as hypersensitivity to dithiothreitol and to benzylpenicillin. In addition, unlike the rest of the dsb genes, dsbD is essential for bacterial growth at temperatures above 42 degrees C. Cloning of the wild-type gene and sequencing and overexpression of the protein show that dsbD is part of an operon and encodes an inner membrane protein. A 138 amino acid subdomain of the protein was purified and shown to possess an oxido-reductase activity in vitro. Expressing this subdomain in the periplasmic space helped restore the phenotypic defects associated with a dsbD null mutation. Interestingly, this domain shares 45% identity with the portion of the eukaryotic protein disulfide isomerase carrying the active site. We further show that in dsbD mutant bacteria the dithiol active sites of DsbA and DsbC proteins are mostly oxidized, as compared with wild-type bacteria. Our results argue that DsbD generates a reducing source in the periplasm, which is required for maintaining proper redox conditions. The finding that overexpression of DsbD leads to a Dsb- phenotype, very similar to that exhibited by dsbA null mutants, is in good agreement with such a model.

Kinetic studies on benzyl alcohol dehydrogenase encoded by TOL plasmid pWWO. A member of the zinc-containing long chain alcohol dehydrogenase family.

The nucleotide sequence of the structural gene for benzyl alcohol dehydrogenase encoded by TOL plasmid pWWO of Pseudomonas putida has been determined. Benzyl alcohol dehydrogenase is a member of the long-chain zinc alcohol dehydrogenase family and, like other alcohol dehydrogenases of this family, contains two zinc atoms per subunit. Benzyl alcohol dehydrogenase, while sharing 31% identical residues with horse liver alcohol dehydrogenase, contains several amino acid substitutions near the active site, some of which may be responsible for the substrate specificity of benzyl alcohol dehydrogenase, which oxidizes exclusively aromatic substrates. Benzyl alcohol dehydrogenase also notably lacks the His51 residue of horse liver alcohol dehydrogenase. Contrary to the results obtained with a mutant human liver alcohol dehydrogenase lacking this residue, the concentration and pKa of solvent proton acceptors had no effect on the catalytic efficiency of benzyl alcohol dehydrogenase. The electronic nature of substituents on the aromatic ring of the substrate influenced the kcat of the enzyme in low concentrations of external proton acceptor, but not in high concentrations. Product inhibition studies demonstrated that benzyl alcohol dehydrogenase followed a general Ordered Bi Bi kinetic mechanism in low proton acceptor conditions, while following a Theorell-Chance kinetic mechanism at high proton acceptor conditions.

Substrate-specificity of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase encoded by TOL plasmid pWW0. Metabolic and mechanistic implications.

The substrate-specificities of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, encoded by TOL plasmid pWW0 of Pseudomonas putida mt-2, were determined. The rates of benzyl alcohol dehydrogenase-catalysed oxidation of substituted benzyl alcohols and reduction of substituted benzaldehydes were independent of the electronic nature of the substituents at positions 3 and 4. Substitutions at position 2 of benzyl alcohol affected the reactivity of benzyl alcohol dehydrogenase: the velocity of the benzyl alcohol dehydrogenase-catalysed oxidation was lower for compounds possessing electron-withdrawing substitutions. In the reverse reaction of benzyl alcohol dehydrogenase, none of the substitutions tested influenced the apparent kcat. values. The rates of benzaldehyde dehydrogenase-catalysed oxidation of substituted benzaldehydes were influenced by the electronic nature of the substitutions: electron-withdrawing groups at positions 3 and 4 favoured the oxidation of benzaldehydes. Substitution at position 2 of benzaldehyde greatly diminished the benzaldehyde dehydrogenase-catalysed oxidation. Substitution at position 2 with electron-donating groups essentially abolished reactivity, and only substitutions that were strongly electron-withdrawing, such as nitro and fluoro groups, permitted enzyme-catalysed oxidation. The influence of the electronic nature and the position of substitutions on the aromatic ring of the substrate on the velocity of the catalysed reactions provided some indications concerning the transition state during the oxidation of the substrates, and on the rate-limiting steps of the enzymes. Pseudomonas putida mt-2 containing TOL plasmid pWW0 cannot grow on toluene derivatives substituted at position 2, nor can it grow on 2-substituted benzyl alcohols or aldehydes. One of the reasons for this may be the substrate-specificities of the benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase.