Chun-Gyu Kim

Neocarzinostatin naphthoate synthase: an unique iterative type I PKS from neocarzinostatin producer Streptomyces carzinostaticus

Enediyne antibiotics are known for their potent antitumor activities. One such enediyne, neocarzinostatin (NCS), consists of a 1:1 complex of non‐peptide chromophore (1a), and peptide apoprotein. The structurally diverse non‐peptide chromophore is responsible for its biological activity. One of its structural components, the naphthoic acid moiety (2,7‐dihydroxy‐5‐methyl‐1‐naphthoic acid, 1d) is synthesized by a polyketide synthase (PKS) pathway through condensing six intact acetate units. The 5.45 kb iterative type I PKS, neocarzinostatin naphthoate synthase (NNS), responsible for naphthoic acid moiety biosynthesis, shares sequence homology with 6‐methyl salicylic acid synthase of fungi and orsellinic acid synthases (AviM and CalO5) of Streptomyces origin. Cultures of S. lividans TK24 and S. coelicolor YU105 containing plasmids with NNS were able to produce 2‐hydroxy‐5‐methyl‐1‐naphthoic acid (2a), a key intermediate of naphthoic acid moiety in NCS. In addition to 2a, a novel product, 2‐hydroxy‐5‐hydroxymethyl‐1‐naphthoic acid (2d) was isolated. This is the first report of a bacterial iterative type I PKS from an enediyne producer which enables the biosynthesis of bicyclic aromatic compounds.

Escherichia coli 6‐pyruvoyltetrahydropterin synthase ortholog encoded by ygcM has a new catalytic activity for conversion of sepiapterin to 7,8‐dihydropterin

The putative gene (ygcM) of Escherichia coli was verified in vitro to encode the ortholog of 6‐pyruvoyltetrahydropterin synthase (PTPS). Unexpectedly, the enzyme was found to convert sepiapterin to 7,8‐dihydropterin without any cofactors. The enzymatic product 7,8‐dihydropterin was identified by HPLC and mass spectrometry analyses, suggesting a novel activity of the enzyme to cleave the C6 side chain of sepiapterin. The optimal activity occurred at pH 6.5–7.0. The reaction rate increased up to 3.2‐fold at 60–80°C, reflecting the thermal stability of the enzyme. The reaction required no metal ion and was activated slightly by low concentrations (1–5 mM) of EDTA. The apparent K m value for sepiapterin was determined as 0.92 mM and the V max value was 151.3 nmol/min/mg. The new catalytic function of E. coli PTPS does not imply any physiological role, because sepiapterin is not an endogenous substrate of the organism. The same activity, however, was also detected in a PTPS ortholog of Synechocystis sp. PCC 6803 but not significant in Drosophila and human enzymes, suggesting that the activity may be prevalent in bacterial PTPS orthologs.

Functional characterization of ketoreductase (rubN6) and aminotransferase (rubN4) genes in the gene cluster of Streptomyces achromogenes var. rubradiris.

ORF’s for rubN6 and rubN4 have been annotated as thymidine diphosphate glucose 4-ketoreductase and thymidine diphosphate glucose 3-aminotransferase by sequence analysis of the rubradirin biosynthetic gene cluster cloned from Streptomyces achromogenes var. rubradiris NRRL 3061. Both ORFs were heterologously expressed in Escherichia coli as His-tagged fusion proteins. The functionalities of TDP-glucose 4-ketoreductase and TDP-glucose 3-aminotransferase were verified by in vitro enzyme assay, and a biosynthetic pathway for TDP-d-rubranitrose is proposed.

Structure Determination of Macrolactin Compounds with Antibacterial Activities Isolated from Bacillus polyfermenticus KJS-2

In this study, we isolated five macrolactin compounds from a fermentation broth of Bacillus polyfermenticus KJS-2. The macrolactin compounds were structurally identified as macrolactin A (MA), 7-O-malonyl macrolactin A (MMA), 7-O-succinyl macrolactin A (SMA), macrolactin E (ME) and macrolactin F (MF) via a variety of NMR techniques, COZY, DEPT, HMQC and HMBC, and mass and specific rotation assays. The three macrolactin compounds, MA, MMA and SMA, profoundly inhibited the growth of both vancomycin-resistant Enterococci (VREs) and methicillin-resistant Staphylococcus aureus (MRSA), the inhibition of which were estimated via a disc agar diffusion bioassay. MA, MMA, and SMA exhibited antibacterial activities superior to those of vancomycin and teicoplanin.

Relative bioavailability of two 5-mg montelukast sodium chewable tablets: a single dose, randomized, open-label, 2-period crossover comparison in healthy korean adult male volunteers.

Montelukast sodium, cysteinyl leukotriene receptor 1 specific antagonist, has been marketed in Korea for the treatment of bronchial asthma and allergic rhinitis. The aim of this study was to compare the pharmacokinetics and relative bioavailability of a test and reference formulation of montelukast 5-mg chewable tablets in healthy Korean male volunteers to meet KFDA regulatory criteria for marketing of the new generic formulation. This study was designed as a single-dose, 2-treatment, and 2-period crossover trial with 32 healthy volunteers. Each subject was randomly assigned to receive the test (Dong-Kook Montelukast Sodium Chewable Tablet 5 mg®) or reference (Singulair Chewable Tablet 5 mg®) formulation. The tablet was chewed 20 times, and then swallowed with 240 mL of water. Plasma concentrations of montelukast up to 24 h after the dose were determined using a validated UPLC-MS/MS method, and the bioequivalence between the 2 formulations was assessed by statistical analysis of mean ratios of log-transformed AUC0-24 h and Cmax. No period or sequence effects were detected. The AUC0-24 h was 1 835 ng·h/mL for the test formulation, and 1 930 ng·h/mL for the reference formulation. The respective values of AUC0-∞ were 1 917 and 2 015 ng·h/mL. The Cmax of the test and reference products (247 and 283 ng/mL, respectively) reached at 2.25 and 2.72 h, respectively. Then, they gradually decreased with the mean terminal t1/2 of 5.25 and 5.30 h for the test and reference products, respectively. The 90% CIs for the ratio of log-transformed AUC0-24 h and Cmax for the test and reference formulations were 0.92-0.99 and 0.83-0.91, respectively. No adverse events were reported in this study. This single dose study found that the test and reference products met the regulatory criteria for bioequivalence in these fasting healthy Korean male volunteers.