Ca(2+)-induced folding of a family I.3 lipase with repetitive Ca(2+) binding motifs at the C-terminus.

In order to understand a role of the Ca(2+) ion on the structure and function of a Ca(2+)-dependent family I.3 lipase from Pseudomonas sp. MIS38, apo-PML, holo-PML, holo-PML*, and the N-terminal domain alone (N-fragment) were prepared and biochemically characterized. Apo-PML and holo-PML represent refolded proteins in the absence and presence of the Ca(2+) ion, respectively. Holo-PML* represents a holo-PML dialyzed against 20 mM Tris-HCl (pH 7.5). The results suggest that the C-terminal domain of PML is almost fully unfolded in the apo-form and its folding is induced by Ca(2+) binding. The folding of this C-terminal domain may be required to make a conformation of the N-terminal catalytic domain functional.

The earliest stages of B cell development require a chemokine stromal cell-derived factor/pre-B cell growth-stimulating factor.

Environmental factors essential for the first stages of B lymphopoiesis remain elusive. Here, we report that immediately after commitment to B lineage, precursors become dependent on a chemokine SDF-1 and its receptor CXCR4 using mutant and radiation chimeric mice. In bone marrow, generation of the earliest identifiable B cell precursor populations requires CXCR4. In fetal liver, we identified Lin(-)CD19(-)c-kit(+)IL-7Ralpha(+)AA4.1(+), the earliest unipotent B cell precursor population, and found that its development was severely affected in SDF-1(-/-) embryos but not in IL-7(-/-) embryos. Lin(-) T cell progenitors appeared normal in SDF-1(-/-) embryos. Moreover, SDF-1 exhibited specific biologic activities on the earliest B cell precursors. SDF-1 provides the first example of a cytokine responsible for the earliest B lineage stages.

Identification of the histidine and aspartic acid residues essential for enzymatic activity of a family I.3 lipase by site-directed mutagenesis.

A lipase from Pseudomonas sp. MIS38 (PML) is a member of the lipase family I.3. We analyzed the roles of the five histidine residues (His(30), His(274), His(291), His(313), and His(365)) and five acidic amino acid residues (Glu(253), Asp(255), Asp(262), Asp(275), and Asp(290)), which are fully conserved in the amino acid sequences of family I.3 lipases, by site-directed mutagenesis. We showed that the mutation of His(313) or Asp(255) to Ala almost fully inactivated the enzyme, whereas the mutations of other residues to Ala did not seriously affect the enzymatic activity. Measurement of the far- and near-UV circular dichroism spectra suggests that inactivation by the mutation of His(313) or Asp(255) is not due to marked changes in the tertiary structure. We propose that His(313) and Asp(255), together with Ser(207), form a catalytic triad in PML.

Overproduction in Escherichia coli, purification and characterization of a family I.3 lipase from Pseudomonas sp. MIS38.

Determination of the nucleotide sequence of the gene encoding a lipase from Pseudomonas sp. MIS38 (PML) revealed that PML is a member of the lipase family I.3 and is composed of 617 amino acid residues with a calculated molecular weight of 64510. Recombinant PML (rPML) was overproduced in Escherichia coli in an insoluble form, solubilized in the presence of 8 M urea, purified in a urea-denatured form and refolded by removing urea in the presence of the Ca(2+) ion. Gel filtration chromatography suggests that this refolded protein is monomeric. rPML showed relatively broad substrate specificities and hydrolyzed glyceryl tributyrate and olive oil with comparable efficiencies. rPML was active only in the form of a holo-enzyme, in which at least 12 Ca(2+) ions bound. These Ca(2+) ions bound too tightly to be removed from the protein upon dialysis, but were removed from it upon EDTA treatment. The resultant apo-enzyme was fully active in the presence of 10 mM CaCl(2), but was inactive in the absence of the Ca(2+) ion. PML has a GXSXG motif, which is conserved in lipases/esterases and generally contains the active-site serine. The mutation of Ser(207) within this motif to Ala completely inactivated PML, suggesting that Ser(207) is the active-site serine of PML.

Identification of the gene encoding esterase, a homolog of hormone-sensitive lipase, from an oil-degrading bacterium, strain HD-1.

The gene encoding an esterase (HDE) was cloned from an oil-degrading bacterium, strain HD-1. HDE is a member of the hormone-sensitive lipase family and composed of 317 amino acid residues with a molecular weight of 33,633. The HDE-encoding gene was expressed in Escherichia coli, and the recombinant protein was purified and characterized. Amino acid sequence analysis indicated that the methionine residue was removed from its NH(2)-terminus. The good agreement of the molecular weights estimated by SDS-PAGE (35,000) and gel filtration (38,000) suggests that it acts in a monomeric form. HDE showed hydrolytic activity towards p-nitrophenyl esters of fatty acids with an acyl chain length of 2 to 14 and tributyrin, whereas it showed little hydrolytic activity towards p-nitrophenyl oleate (C(18)), tricaprylin and triolein. Determination of the kinetic parameters for the hydrolyses of the p-nitrophenyl substrates from C(2) to C(14) indicated that HDE shows a relatively broad substrate specificity. However, comparison of the k(cat)/K(m) values indicated that the C(10)-C(14) substrates are the most preferred ones. Such a preference for substrates with long acyl chains may be a characteristic of HDE.

Gene cloning and characterization of aldehyde dehydrogenase from a petroleum-degrading bacterium, strain HD-1.

The hd-ald gene encoding aldehyde dehydrogenase (hd-ALDH) from an mixotrophic petroleum-degrading bacterium, strain HD-1 was cloned and sequenced. hd-ALDH (506 amino acids) is a member of the NAD+-dependent aldehyde dehydrogenase group. The hd-ald gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized biochemically and enzymatically. The molecular weight of the enzyme was estimated to be 55,000 by SDS-PAGE, and 224,000 by gel filtration chromatography, suggesting that it acts as a tetramer. The CD spectrum suggests that the helical content of the enzyme is 10%. hd-ALDH was active on various aliphatic aldehyde substrates. The K(m) values of the enzyme were 6.4 microM for acetaldehyde, 4.2 microM for hexanal, 2.8 microM for octanal, and 0.84 microM for decanal, whereas the kcat values for these substrates were nearly equal (51-64 min(-1)). These results indicate that hd-ALDH acts preferentially on long-chain aliphatic aldehydes.

Characterization of petroleum-degrading bacteria from oil-contaminated sites in Vietnam.

Four petroleum-degrading bacterial strains, 2TN-NB, 6TBX-CL, MVK2-5, and XCK, were isolated from various oil-contaminated sites in Vietnam. Determination of the nucleotide sequence of the gene encoding 16S rRNA allowed 2TN-NB to be identified as Acinetobacter sp. and the other three stains as Pseudomonas sp. Among the four isolates, 2TN-NB was most effective in degrading crude oil: in 1 d, it degraded 95% of the crude oil in the culture medium (5%, v/v). The isolated strains could also degrade a sulfur-containing aromatic hydrocarbon, dibenzothiophene (DBT), with low efficiency. Except for MVK2-5, which degraded crude oil least efficiently, the isolates produced biosurfactants in amounts sufficient for structural analysis. FT-IR measurement suggested that strains 6TBX-CL and XCK produced glycolipid-type biosurfactants while that produced by 2TN-NB was of the polysaccharide type.

ATP-, K+-dependent heptamer exchange reaction produces hybrids between GroEL and chaperonin from Thermus thermophilus.

Chaperonin from Thermus thermophilus (Tcpn6014.Tcpn107) splits at the plane between two Tcpn607 rings into two parts in a solution containing ATP and K+ (Ishii, N., Taguchi, H., Sasabe, H., and Yoshida, M. (1995) FEBS Lett. 362, 121-125). When Escherichia coli GroEL14 was additionally included in the solution described above, hybrid chaperonins GroEL7.Tcpn607 and GroEL7. Tcpn607.Tcpn107 were formed rapidly (<20 s) at 37 degrees C. The hybrid was also formed from Tcpn6014 and GroEL14 but not from a mutant GroEL14 lacking ATPase activity. The hybrid formation was saturated at approximately 300 microM ATP and approximately 300 mM K+. These results imply that GroEL14 also splits and undergoes a heptamer exchange reaction with Thermus chaperonin under nearly physiological conditions. Similar to parent chaperonins, the isolated hybrid chaperonins exhibited ATPase activity that was susceptible to inhibition by Tcpn107 or GroES7 and mediated folding of other proteins. Once formed, the hybrid chaperonins were stable, and the parent chaperonins were not regenerated from the isolated hybrids under the same conditions in which the hybrids had been formed. Only under conditions in which GroEL in the hybrids was selectively destroyed, such as incubation at 70 degrees C, Thermus chaperonin, but not GroEL14, was regenerated from the hybrid. Therefore, the split reaction may not be an obligatory event repeated in each turnover of the chaperonin functional cycles but an event that occurs only when chaperonin is first exposed to ATP/K+.

Chaperonin-mediated folding of green fluorescent protein.

Chaperonin-mediated folding of green fluorescent protein (GFP) was examined by real-time monitoring of recovery of fluorescence and by gel filtration high-performance liquid chromatography. Acid-denatured GFP can fold spontaneously upon dilution into the neutral buffer. When Escherichia coli GroEL/ES was present, folding of GFP was arrested. Folding was resumed by subsequent addition of 100 microM or 1 mM ATP, and native GFP was regenerated to 100% yield. When folding was resumed by 10 microM ATP (1.4 mol/mol GroEL subunit), about 60% of GFP recovered native structure, and one-half of them (30%) was found to be still bound to GroEL/ES, indicating the occurrence of folding in the central cavity of the GroEL ring underneath GroES (cis-folding). Because the overall rates of GroEL/ES-, ATP-mediated GFP folding were all similar to that of spontaneous folding, it was concluded that cis-folding proceeded as fast as spontaneous folding. The GroEL/ES-bound native GFP was observed only when both GroES and ATP (but not ADP) were present in the folding mixture. Holo-chaperonin from Thermus thermophilus, which was purified as a cpn60/10 complex, exhibited the similar cis-folding. Consistently, ATP-dependent exchange of cpn10 in the holo-chaperonin with free cpn10 was observed.

Molecular cloning, expression, and characterization of chaperonin-60 and chaperonin-10 from a thermophilic bacterium, Thermus thermophilus HB8.

The gene coding a chaperonin from a thermophilic bacterium, Thermus thermophilus HB8, was cloned and sequenced. The operon structure was the same as those of other bacterial chaperonins and the deduced amino acid sequences of both subunits were highly homologous to those of other chaperonins. The cloned genes of chaperonin subunits, chaperonin-10 (T.th cpn10) and chaperonin-60 (T.th cpn60), were separately expressed in Escherichia coli cells. The expressed subunits were easily purified from other host proteins including GroE, a chaperonin of E. coli. T.th cpn60 was expressed as a tetradecameric form, like GroEL of E. coli. Since chaperonin from T. thermophilus HB8 is purified as a holochaperonin, a complex of tetradecameric T.th cpn60 and heptameric T.th cpn10, a tetradecamer of T.th cpn60 without T.th cpn10 has not been obtained before. T.th cpn60 tetradecamer tended to dissociate into monomers during storage. T.th cpn10 expressed in E. coli was purified as a stable oligomer, most likely a heptamer. The activity as holo-chaperonin was reconstituted by mixing both subunits. T.th cpn60 tetradecamer itself arrested refolding of other proteins. The monomerized T.th cpn60 was easily purified from T.th cpn60 oligomer by gel permeation chromatography. Thus-obtained T.th cpn60 monomer had an ATP-independent chaperone activity, as shown for T.th cpn60 monomer isolated from authentic holo-chaperonin.