Yasuhiro Isogai

Efficient synthesis of optically pure alcohols by asymmetric hydrogen-transfer biocatalysis: application of engineered enzymes in a 2-propanol–water medium

We describe an efficient method for producing both enantiomers of chiral alcohols by asymmetric hydrogen-transfer bioreduction of ketones in a 2-propanol (IPA)–water medium with E. coli biocatalysts expressing phenylacetaldehyde reductase (PAR: wild-type and mutant enzymes) from Rhodococcus sp. ST-10 and alcohol dehydrogenase from Leifsonia sp. S749 (LSADH). We also describe the detailed properties of mutant PARs, Sar268, and HAR1, which were engineered to have high activity and productivity in media composed of polar organic solvent and water, and the construction of three-dimensional structure of PAR by homology modeling. The Km and Vmax values for some substrates and the substrate specificity of mutant PARs were quite different from those of wild-type PAR. The results well explained the increased productivity of engineered PARs in IPA–water medium.

The Transmembrane Region of Guard Cell SLAC1 Channels Perceives CO2 Signals via an ABA-Independent Pathway in Arabidopsis.

A transmembrane region of the guard cell SLAC1 anion channel contains sites of CO2 signal perception, which could be different from the sites of ABA signal perception. The guard cell S-type anion channel, SLOW ANION CHANNEL1 (SLAC1), a key component in the control of stomatal movements, is activated in response to CO2 and abscisic acid (ABA). Several amino acids existing in the N-terminal region of SLAC1 are involved in regulating its activity via phosphorylation in the ABA response. However, little is known about sites involved in CO2 signal perception. To dissect sites that are necessary for the stomatal CO2 response, we performed slac1 complementation experiments using transgenic plants expressing truncated SLAC1 proteins. Measurements of gas exchange and stomatal apertures in the truncated transgenic lines in response to CO2 and ABA revealed that sites involved in the stomatal CO2 response exist in the transmembrane region and do not require the SLAC1 N and C termini. CO2 and ABA regulation of S-type anion channel activity in guard cells of the transgenic lines confirmed these results. In vivo site-directed mutagenesis experiments targeted to amino acids within the transmembrane region of SLAC1 raise the possibility that two tyrosine residues exposed on the membrane are involved in the stomatal CO2 response.

Dominant and recessive mutations in the Raf-like kinase HT1 gene completely disrupt stomatal responses to CO2 in Arabidopsis

Highlight Loss-of-function and gain-of-function ht1 Arabidopsis mutants have completely disrupted CO2 responses due to reduced and enhanced kinase activities, respectively.

Effect of pressure on the secondary structure of coiled coil peptide GCN4-p1.

It has recently been demonstrated that pressure induces folding of the alpha-helix of an alanine-based peptide (AK20), which is a monomer in water (Imamura and Kato, Proteins 2009;76:911-918). The present study focused on a coiled coil peptide GCN4-p1, the alpha-helices of which associate via a hydrophobic core, to examine whether the pressure stability of the alpha-helices depends on the hydrophobic core. Fourier transform infrared spectroscopy was used to investigate the effect of pressure on the secondary structures of GCN4-p1. The infrared spectra of GCN4-p1 shows the two amide I peaks at approximately 1650 and approximately 1630 cm(-1) stemming from the solvent-inaccessible alpha-helix and the solvent-accessible alpha-helix, respectively. The intensities of both the peaks increase with increasing pressure, whereas they decrease with increasing temperature. This indicates that pressure induces both the alpha-helices of GCN4-p1 to fold. The present result suggests that the positive volume change upon unfolding of an alpha-helix is a common characteristic of peptides. The pressure-induced stabilization of the alpha-helices is discussed in comparison with the pressure denaturation of proteins.

Agrobacterium tumefaciens-mediated transformation of antifungal lipopeptide producing fungus Coleophoma empetri F-11899

The filamentous fungus Coleophoma empetri F-11899 produces an echinocandin-like compound FR901379, the original source for micafungin which is prescribed to treat deep-seated mycoses. Despite its industrial importance, no genetic information on C. empetri F-11899 is currently available. To characterize FR901379 biosynthetic genes by insertional mutagenesis and to improve the compound production genetically, Agrobacterium tumefaciens-mediated transformation (ATMT) was attempted to make genetic manipulation possible in this strain. The optimum conditions for ATMT of C. empetri were determined for the cell density of bacteria, time period of co-cultivation and types of filters in co-cultivation. Using the established ATMT method, the hygromycin B resistant gene was successfully transferred into the genome of C. empetri F-11899 and stably maintained even after a serial passage. Some of these results will be applicable for ATMT of various filamentous fungi.

Strain selection and scale-up fermentation for FR901379 acylase production by Streptomyces sp. no. 6907.

Micafungin (FK463) is a widely used treatment for life-threatening, deep-seated fungal infections. It is an echinocandin-like lipopeptide derived from the chemical modification of deacylated FR901379, a type of lipopeptide antibiotic produced by Coleophoma empetri F-11899. The palmitoyl moiety of FR901379 is deacylated by FR901379 acylase produced by Streptomyces sp. no. 6907. In this study, our goal was to generate an improved strain of Streptomyces sp. no. 6907 capable of hyperproducing the FR901379-acylase enzyme. To accomplish this goal, modified strains of Streptomyces sp. no. 6907 were generated using UV-irradiation mutagenesis, and strain selection was performed using an agar-plate screening method to efficiently select an acylase-hyperproducing strain. Three marker indices were shown to correlate with elevated acylase production: decreased candidacidal activity of FR901379, decreased proteolytic activity on skim milk, and phenotypic characteristics. Cloning and subsequent sequencing of the acylase gene from the hyperproducing mutant revealed no mutations in either the acylase structural gene or the 5-flanking region required for gene expression. The growth medium was also modified to maximize acylase production. We successfully increased acylase activity approximately 65-fold, compared with the original growth conditions (wild strain cultured in the original unmodified medium). To minimize formation of excess foam during the fermentation process, we optimized the parameters of agitation speed, as calculated from the discharge flow rate. Using our improved strain and the optimized medium and growth conditions, we have developed an improved and highly reproducible method for stable large-scale production of FR901379-acylase.

Modulation of the intermolecular interaction of myoglobin by removal of the heme.

The present study analysed small-angle X-ray scattering profiles of myoglobin to examine how removal of the heme changes the intermolecular interaction.

Alteration of substrate selection of antibiotic acylase from β-lactam to echinocandin

The antibiotic acylases belonging to the N-terminal nucleophile hydrolase superfamily are key enzymes for the industrial production of antibiotic drugs. Cephalosporin acylase (CA) and penicillin G acylase (PGA) are two of the most intensively studied enzymes that catalyze the deacylation of β-lactam antibiotics. On the other hand, aculeacin A acylase (AAC) is known to be an alternative acylase class catalyzing the deacylation of echinocandin or cyclic lipopeptide antibiotic compounds, but its structural and enzymatic properties remain to be explored. In the present study, 3D homology models of AAC were constructed, and docking simulation with substrate ligands was performed for AAC, as well as for CA and PGA. The docking models of AAC with aculeacin A suggest that AAC has the deep narrow binding pocket for the long-chain fatty acyl group of the echinocandin molecule. To confirm this, CA mutants have been designed to form the binding pocket for the long acyl chain. Experimentally synthesized mutant enzymes exhibited lower enzymatic activity for cephalosporin but higher activity for aculeacin A, in comparison with the wild-type enzyme. The present results have clarified the difference in mechanisms of substrate selection between the β-lactam and echinocandin acylases and demonstrate the usefulness of the computational approaches for engineering the enzymatic properties of antibiotic acylases.

Supramolecular polymer formation by a de novo hemoprotein with a synthetic diheme compound

Proteins are attractive materials for supramolecular chemistry due to their multifunctionality and self‐organization ability. In this work, we synthesized a diheme compound, in which two iron‐protoporphyrin IX molecules are associated via a linker chain, and introduced it into a de novo designed four‐helix bundle protein with two heme‐binding sites. The protein gradually bound the diheme compound by bis‐histidyl ligation and formed supramolecular polymers. Polymer formation was observed by atomic force microscopy (AFM), which revealed the highly branched, dendritic forms of the fibrous architecture. The present results may open a pathway toward nanowire construction with de novo heme‐proteins.

Functional Characterization of Epitheaflagallin 3-O-Gallate Generated in Laccase-Treated Green Tea Extracts in the Presence of Gallic Acid

Epitheaflagallin (ETFG) and epitheaflagallin 3-O-gallate (ETFGg) are minor polyphenols in black tea extract that are enzymatically synthesized from epigallocatechin (EGC) and epigallocatechin gallate (EGCg), respectively, in green tea extract via laccase oxidation in the presence of gallic acid. The constituents of laccase-treated green tea extract in the presence of gallic acid are thus quite different from those of nonlaccase-treated green tea extract: EGC and EGCg are present in lower concentrations, and ETFG and ETFGg are present in higher concentrations. Additionally, laccase-treated green tea extract contains further polymerized catechin derivatives, comparable with naturally fermented teas such as oolong tea and black tea. We found that ETFGg and laccase-treated green tea extracts exhibit versatile physiological functions in vivo and in vitro, including antioxidative activity, pancreatic lipase inhibition, Streptococcus sorbinus glycosyltransferase inhibition, and an inhibiting effect on the activity of matrix metalloprotease-1 and -3 and their synthesis by human gingival fibroblasts. We confirmed that these inhibitory effects of ETFGg in vitro match well with the results obtained by docking simulations of the compounds with their target enzymes or noncatalytic protein. Thus, ETFGg and laccase-treated green tea extracts containing ETFGg are promising functional food materials with potential antiobesity and antiperiodontal disease activities.