Yen-Chung Lee

Synergistic activities of tigecycline with clarithromycin or amikacin against rapidly growing mycobacteria in Taiwan

The occurrence of diseases caused by rapidly growing mycobacteria (RGM) is increasing in Taiwan. In this study, the in vitro antimicrobial activities of tigecycline, minocycline, tetracycline and doxycycline were evaluated against 160 clinical RGM isolates, including 34 Mycobacterium abscessus sensu stricto (s.s.), 44 Mycobacterium massiliense, 1 Mycobacterium bolletii, 58 Mycobacterium fortuitum and 23 Mycobacterium chelonae. Clarithromycin and amikacin were tested alone as well as for synergistic effect with tigecycline. Both amikacin and tigecycline showed excellent activities against the RGM. More than 85% of each of the five RGM species isolates showed susceptibility to the two drugs. The MIC₅₀ and MIC₉₀ values (drug concentrations at which 50% and 90%, respectively, of the tested isolates did not show any visible growth) of amikacin were 1-4 mg/L and 2-8 mg/L, respectively, whilst those of tigecycline were 0.125-1 mg/L and 0.5-2.0 mg/L. Clarithromycin had only moderate activity, with ≥42.9% but ≤87.5% of each RGM species isolates showing susceptibility. The other three drugs had limited or no antimicrobial activity, with <40% of each RGM species isolates showing susceptibility. Combined with clarithromycin, tigecycline had synergistic activity against 92.9%, 68.8%, 100%, 35.7% and 46.2% of M. abscessus s.s., M. massiliense, M. bolletii, M. fortuitum and M. chelonae isolates, respectively. However, tigecycline combined with amikacin had synergistic activity against <25% but antagonistic activity against >18% of each RGM species. Thus, tigecycline alone may be an alternative for treating RGM diseases in patients who are intolerant to cefoxitin, imipenem or amikacin. However, it should be used with caution or not used in combination with amikacin for RGM diseases.

Characterization of glycine substitution mutations within the putative NAD+-binding site of Bacillus licheniformis aldehyde dehydrogenase.

The NAD(+)-requiring enzymes of the aldehyde dehydrogenase (ALDH) family contain a glycine motif, GX1- 2GXXG, which is reminiscent of the fingerprint region of the Rossman fold, a conserved structural motif of the classical nicotinamide nucleotide-binding proteins. In this research, the role of three glycine residues situated within the putative NAD(+)-binding motif (211-GPGSSAG) together with Gly233 and Gly238 of Bacillus licheniformis ALDH (BlALDH) were probed by site-directed mutatgenesis. Fifteen mutant BlALDHs were obtained by substitution of the indicated glycine residues with alanine, glutamate and arginine. Except for the Ala replacement at positions 211, 213, 217 and 238, the remaining mutant enzymes lost the dehydrogenase activity completely. Tryptophan fluorescence and far-UV circular dichroism spectra allowed us to discriminate BlALDH and the inactive mutant enzymes, and unfolding analyses further revealed that they had a different sensitivity towards temperature- and guanidine hydrochloride (GdnHCl)-induced denaturation. BlALDH and the functional variants had a comparable T(m) value, but the value was reduced by more than 5.1°C in the rest of mutant enzymes. Acrylamide quenching analysis showed that the inactive mutant enzymes had a dynamic quenching constant greater than that of BlALDH. Native BlALDH started to unfold beyond ~0.21 M GdnHCl and reached an unfolded intermediate, [GdnHCl](0.5, N-U), at 0.92 M equivalent to free energy change (ΔG(N-U)(H2O)) of 12.34 kcal/mol for the N → U process, whereas the denaturation midpoints for mutant enzymes were 0.45-1.61 M equivalent to ΔG(N-U)(H2O) of 0.31-4.35 kcal/mol. Taken together, these results strongly suggest that the explored glycines are indeed important for the catalytic activity and structural stability of BlALDH.

Contribution of conserved Glu255 and Cys289 residues to catalytic activity of recombinant aldehyde dehydrogenase from Bacillus licheniformis

Based on the sequence homology, we have modeled the three-dimensional structure of Bacillus licheniformis aldehyde dehydrogenase (BlALDH) and identified two different residues, Glu255 and Cys289, that might be responsible for the catalytic function of the enzyme. The role of these residues was further investigated by site-directed mutagenesis and biophysical analysis. The expressed parental and mutant proteins were purified by nickel-chelate chromatography, and their molecular masses were determined to be approximately 53 kDa by SDS-PAGE. As compared with the parental BlALDH, a dramatic decrease or even complete loss of the dehydrogenase activity was observed for the mutant enzymes. Structural analysis showed that the intrinsic fluorescence and circular dichroism spectra of the mutant proteins were similar to the parental enzyme, but most of the variants exhibited a different sensitivity towards thermal- and guanidine hydrochloride-induced denaturation. These observations indicate that residues Glu255 and Cys289 play an important role in the dehydrogenase activity of BlALDH, and the rigidity of the enzyme has been changed as a consequence of the mutations.

Biocatalytic Synthesis of γ-glutamyl-L-leucine, a Kokumi-Imparting Dipeptide, by Bacillus licheniformis γ-Glutamyltranspeptidase

ABSTRACT Several γ-glutamyl compounds are known to have favorable features for a variety of biotechnological and pharmaceutical applications. However, so far only a limited number of γ-glutamyl compounds have been synthesized through γ-glutamyltranspeptidase (GGT). With the help of thin layer chromatography and computer-assisted image analysis techniques, we performed an enzymatic method involving Bacillus licheniformis GGT (BlGGT) for the synthesis of γ-glutamyl-L-leucine (γ-Glu-Leu). The optimum conditions for a biocatalytic synthesis of γ-Glu-Leu were determined to be 200 mM glutamine, 200 mM leucine, 50 mM Tris-HCl buffer (pH 9.0), and BlGGT at a working concentration of 2.0 U/mL. Upon a 5-h incubation of the reaction mixture at 50°C, the product yield could reach approximately 52%. The preparative synthesis of γ-Glu-Leu by BlGGT was done under optimal conditions at millimol scale level. The reaction products were subjected to mass spectrometry/nuclear magnetic resonance analyses to ensure the successful synthesis of γ-Glu-Leu. Taken together, the present study demonstrates the high potentiality of BlGGT-catalyzed reaction in the synthesis of γ-glutamyl compounds.