Validamycin B

An ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established for the determination of four highly polar agricultural antibiotics kasugamycin, validamycin A, ningnanmycin, and polyoxin B in plant-derived foods. The samples were extracted with a 0.2% formic acid solution, purified by hydrophilic-lipophilic balance and mixed-mode cation-exchange solid-phase extraction, and then reconstituted for UPLC-MS/MS detection. The chromatographic analysis was performed on a BEH Amide column (100 mm × 2.1 mm, 1.7 μm) using gradient elution with a 0.1% formic acid solution and 0.1% formic acid acetonitrile as mobile phases. Method validation was performed on 15 matrices spiked at 0.02 (or 0.05), 0.5, and 2 mg/kg. The mean recovery rate ranged from 75 to 102% with relative standard deviations (RSD) was less than 20%. Good linearities (r> 0.99) in the range of 0.002-0.2 μg/mL were obtained. The limits of quantification (LOQs) were 0.02 and 0.05 mg/kg. Studies on the stability of the analytes in the stored kiwifruit samples showed that kasugamycin, validamycin A, and ningnanmycin were stable for at least 6 months, while polyoxin B was observed to be partially degraded (the degradation rate at 6 months was 31.3%). The method was demonstrated to be effective and reliable in real samples. In the kiwifruit samples treated after 7 days, no residues of ningnanmycin and polyoxin B were detected, while the residues of kasugamycin and validamycin A were 0.12 and 0.038 mg/kg, respectively.

A reversed-phase high-performance liquid chromatography method for the quantitative analysis of valienamine in the microbial degradation of validamycin A, using a procedure for pre-column derivatization of valienamine with p-nitrofluorobenzene is described. Valienamine in the broth was first isolated with the ion-exchange method. The optimized conditions for the derivatization were the reaction time 30 min and reaction temperature 100 degrees C. With the mobile phases consisting of acetonitrile-water (12:88) (eluent A) and methanol (eluent B), the gradient was carried out with 100% of A for 15 min and then 100% of B for another 10 min. The parameters in the process were the flow rate of the mobile phase 1.0 ml/min, the injection volume 20 microl, the column temperature 40 degrees C and wavelength of ultraviolet detection 398 nm in all runs. A good linearity was found in the range of 0.5-150.0 microg/ml. Both intra- and inter-day precisions of valienamine, expressed as the relative standard deviation, were less than 9.4%. Accuracy, expressed as the relative error, range from -0.5 to 2.7%. The mean absolute recovery of valienamine at three different concentrations was 94.2%. The method was proved suitable for the study on the process of microbial degradation of validamycin A to produce valienamine.

Validamycin A (Val-A) is an effective antifungal agent widely used in Asian countries as crop protectant. Validoxylamine A, the core structure and intermediate of Val-A, consists of two C(7)-cyclitol units connected by a rare C-N bond. In the Val-A biosynthetic gene cluster in Streptomyces hygroscopicus 5008, the ORF valL was initially annotated as a validoxylamine A 7'-phosphate(V7P) synthase, whose encoded 497-aa protein shows high similarity with trehalose 6-phosphate(T6P) synthase. Gene inactivation of valL abolished both validoxylamine A and validamycin A productivity, and complementation with a cloned valL recovered 10% production of the wild-type in the mutant, indicating the involvement of ValL in validoxylamine A biosynthesis. Also we determined the structures of ValL and ValL/trehalose complex. The structural data indicates that ValL adopts the typical fold of GT-B protein family, featuring two Rossmann-fold domains and an active site at domain junction. The residues in the active site are arranged in a manner homologous to that of Escherichia coli (E.coli) T6P synthase OtsA. However, a significant discrepancy is found in the active-site loop region. Also noticeable structural variance is found around the active site entrance in the apo ValL structure while the region takes an ordered configuration upon binding of product analog trehalose. Furthermore, the modeling of V7P in the active site of ValL suggests that ValL might have a similar SNi-like mechanism as OtsA.