Kei Amada

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.

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.

Ca2+-induced folding of a family I.3 lipase with repetitive Ca2+ binding motifs at the C-terminus

In order to understand a role of the Ca2+ ion on the structure and function of a Ca2+‐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 Ca2+ 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 Ca2+ binding. The folding of this C‐terminal domain may be required to make a conformation of the N‐terminal catalytic domain functional.

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 (His30, His274, His291, His313, and His365) and five acidic amino acid residues (Glu253, Asp255, Asp262, Asp275, and Asp290), which are fully conserved in the amino acid sequences of family I.3 lipases, by site‐directed mutagenesis. We showed that the mutation of His313 or Asp255 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 His313 or Asp255 is not due to marked changes in the tertiary structure. We propose that His313 and Asp255, together with Ser207, form a catalytic triad in PML.

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.

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). WhenEscherichia 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 °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 ∼300 μm ATP and ∼300 mmK+. These results imply that GroEL14 also splits and undergoes a heptamer exchange reaction withThermus 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 °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+.