Hiroko Tokunaga

Engineering of halophilic enzymes: Two acidic amino acid residues at the carboxy-terminal region confer halophilic characteristics to Halomonas and Pseudomonas nucleoside diphosphate kinases

Nucleoside diphosphate kinase from Halomonas sp. 593 (HaNDK) exhibits halophilic characteristics. Residues 134 and 135 in the carboxy‐terminal region of HaNDK are Glu–Glu, while those of its homologous counterpart of non‐halophilic Pseudomonas NDK (PaNDK) are Ala–Ala. The double mutation, E134A‐E135A, in HaNDK results in the loss of the halophilic characteristics, and, conversely, the double mutation of A134E‐A135E in PaNDK confers halophilic characters to this enzyme, indicating that the charged state of these two residues that are located in the C‐terminal region plays a critical role in determining halophilic characteristics. The importance of these two residues versus the net negative charges will be discussed in relation to the halophilicity of NDK.

NaCl-activated nucleoside diphosphate kinase from extremely halophilic archaeon, Halobacterium salinarum, maintains native conformation without salt

Enzymes from extremely halophilic archaea are readily denatured in the absence of a high salt concentration. However, we have observed here that a nucleoside diphosphate kinase prepared from Halobacterium salinarum was active and stable in the absence of salt, though it has the amino acid composition characteristic of halophilic enzymes. Recombinant nucleoside diphosphate kinase expressed in Escherichia coli requires salt for activation in vitro, but once it acquires the proper folding, it no longer requires the presence of salts for its activity and stability.

Highly efficient renaturation of β-lactamase isolated from moderately halophilic bacteria

Most, if not all, β‐lactamases reported to date are irreversibly denatured at 60–70°C. Here, we found that a halophilic β‐lactamase from the moderately halophilic bacterium Chromohalobacter sp. 560 was highly stable against heat inactivation: it retained ∼75% of its activity after boiling for 5 min in the presence of 0.2 M NaCl, suggesting that the protein either incompletely denatures during the boiling process or readily renatures upon cooling to the assay temperature. Circular dichroism showed a complete unfolding at 60°C and a full reversibility, indicating that the observed activity after boiling is due to efficient refolding following heat denaturation. The enzyme showed optimal activity at 50–60°C, indicating that an increase in activity with temperature offsets the thermal denaturation. The gene bla was cloned, and the primary structure of the enzyme was deduced to be highly abundant in acidic amino acid residues, one of the characteristics of halophilic proteins. Despite its halophilic nature, the enzyme refolds in low salt media after heat denaturation.

Efficient secretion of human lysozyme from the yeast, Kluyveromyces lactis.

Efficient secretion of human lysozyme from the yeast, Kluyveromyces lactis, was achieved by using more stable vectors in the order of S11 replication origin-containing episomal vector < full-length K. lactis plasmid pKD1-containing vector < centromeric vector < chromosome-integrated vectors. Cells containing a PGK (phosphoglycerate kinase) promoter-driven integration vector grown in non-selective rich medium achieved the highest level of secretion, ∼100 μg lysozyme secretion ml −1 culture: this level was ∼10-fold higher than that achieved by episomal vectors. An additional copy of the protein disulfide isomerase gene further facilitated the secretion.

Salt-Inducible Multidrug Efflux Pump Protein in the Moderately Halophilic Bacterium Chromohalobacter sp.

ABSTRACT It has been known that halophilic bacteria often show natural resistance to antibiotics, dyes, and toxic metal ions, but the mechanism and regulation of this resistance have remained unexplained. We have addressed this question by identifying the gene responsible for multidrug resistance. A spontaneous ofloxacin-resistant mutant derived from the moderately halophilic bacterium Chromohalobacter sp. strain 160 showed a two- to fourfold increased resistance to structurally diverse compounds, such as tetracycline, cefsulodin, chloramphenicol, and ethidium bromide (EtBr), and tolerance to organic solvents, e.g., hexane and heptane. The mutant produced an elevated level of the 58-kDa outer membrane protein. This mutant (160R) accumulated about one-third the level of EtBr that the parent cells did. An uncoupler, carbonyl cyanide m-chlorophenylhydrazone, caused a severalfold increase in the intracellular accumulation of EtBr, with the wild-type and mutant cells accumulating nearly equal amounts. The hrdC gene encoding the 58-kDa outer membrane protein has been cloned. Disruption of this gene rendered the cells hypersusceptible to antibiotics and EtBr and led to a high level of accumulation of intracellular EtBr. The primary structure of HrdC has a weak similarity to that of Escherichia coli TolC. Interestingly, both drug resistance and the expression of HrdC were markedly increased in the presence of a high salt concentration in the growth medium, but this was not observed in hrdC-disrupted cells. These results indicate that HrdC is the outer membrane component of the putative efflux pump assembly and that it plays a major role in the observed induction of drug resistance by salt in this bacterium.

Efficient expression, purification and characterization of mouse salivary α-amylase secreted from methylotrophic yeast, Pichia pastoris

We constructed a secretion vector of mouse salivary α‐amylase, pPAM, using the AOX1 promoter‐terminator and the secretion signal of 128 kDa pGKL killer protein, for an alternative yeast, Pichia pastoris. Taking advantage of multicopy insertion of the expression cassette and optimized growth conditions, we succeeded in highly efficient extracellular production (∼240 µg/ml) of mouse α‐amylase in the 10 ml scale by conventional flask culture: this efficiency was about 90‐fold higher than that of Saccharomyces cerevisiae. Growth temperature of cells was critical for efficient production of α‐amylase. P. pastoris transformants secreted both core‐glycosylated and non‐glycosylated α‐amylase molecules with a glycosylated:non‐glycosylated ratio of about 20:80. Both glycosylated and non‐glycosylated α‐amylases were purified separately to apparent homogeneity. The signal sequence was correctly processed in both species, and the molecular masses of glycosylated and non‐glycosylated α‐amylase were determined to be 58 600 and 56 300, respectively, by mass spectrometry. We further studied the outer chain glycosylation of engineered mouse α‐amylase secreted by P. pastoris. Copyright

SECRETION OF MOUSE ALPHA -AMYLASE FROM KLUYVEROMYCES LACTIS

We constructed two mouse α‐amylase secretion vectors for Kluyveromyces lactis using the well‐characterized signal sequence of the pGKL 128 kDa killer precursor protein. Both PHO5 and PGK expression cassettes from Saccharomyces cerevisiae directed the expression of mouse α‐amylase in YPD medium at a similar level of efficiency. K. lactis transformants secreted glycosylated and non‐glycosylated α‐amylase into the culture medium and both species were enzymatically active. The K. lactis/S. cerevisiae shuttle secretion vector pMI6 was constructed, and K. lactis MD2/1(pMI6) secreted about four‐fold more α‐amylase than S. cerevisiae YNN27 harboring the same plasmid, indicating that K. lactis is an efficient host cell for the secretion and production of recombinant proteins.

Halophilic β-lactamase as a new solubility- and folding-enhancing tag protein: production of native human interleukin 1α and human neutrophil α-defensin

The amino acid composition of halophilic enzymes is characterized by an abundant content of acidic amino acid, which confers to the halophilic enzymes extensive negative charges at neutral pH and high aqueous solubility. This negative charge prevents protein aggregation when denatured and thereby leads to highly efficient protein refolding. β-Lactamase from periplasmic space of moderate halophile (BLA), a typical halophilic enzyme, can be readily expressed as a native, active form in Escherichia coli cytoplasm. Similar to other halophilic enzymes, BLA is soluble upon denaturation by heat or urea treatments and, hence, can be efficiently refolded. Such high solubility and refolding efficiency make BLA a potential fusion partner for expression of aggregation-prone heterologous proteins to be expressed in E. coli. Here, we succeeded in the soluble expression of several “difficult-to-express” proteins as a BLA fusion protein and verified biological activities of human interleukin 1α and human neutrophil α-defensin, HNP-1.

Residue 134 determines the dimer-tetramer assembly of nucleoside diphosphate kinase from moderately halophilic bacteria

Halomonas nucleoside diphosphate kinase (HaNDK) forms a dimeric assembly and Pseudomonas NDK (PaNDK) forms a tetrameric assembly. The mutation of Glu134 to Ala in HaNDK resulted in the conversion of the native dimeric structure to the tetramer assembly. Conversely, the mutation of Ala134 to Glu in PaNDK lead to the conversion from the tetramer to the dimer assembly, indicating that a single amino acid substitution at position 134 results in an alteration of the oligomeric structure of NDK. By modeling the structure of HaNDK and PaNDK based on the crystal structure of Myxococcus NDK, we showed that Glu134 exerts sufficient repulsive forces to disrupt the dimer–dimer interaction and prevent the formation of the tetramer.

Properties of chemically modified porin from Escherichia coli in lipid bilayer membranes

Purified porin OmpF from Escherichia coli outer membrane was chemically modified by acetylation and succinylation of amino groups and by amidation of the carboxyl groups. Native and chemically modified porins were incorporated into lipid bilayer membranes and the permeability properties of the pores were studied. Acetylation and succinylation of the porin trimers had almost no influence on the single channel conductance in the presence of small cations and anions and the cation selectivity remained essentially unchanged as compared with the native porin. Amidation had also only little influence on the single channel conductance and changed the pore conductance at maximum by less than 50%, whereas the cation selectivity of the porin is completely lost after amidation. The results suggest that the structure of the porin pore remains essentially unchanged after chemical modification of the pores and that their cation selectivity is caused by an excess of negatively charged groups inside the pore and/or on the surface of the protein. Furthermore, it seems very unlikely that the pore contains any positively charged group at neutral pH.