Structural and Functional Analysis of Validoxylamine A 7′-phosphate Synthase ValL Involved in Validamycin A Biosynthesis

et al. (2012) Structural and Functional Analysis of Validoxylamine A 79-phosphate Synthase ValL Involved in Validamycin A Biosynthesis. PLoS ONE 7(2): e32033. doi:10.1371/journal.pone.0032033 Structural and Functional Analysis of Validoxylamine A 79-phosphate Synthase ValL Involved in Validamycin A Biosynthesis Lina Zheng 0 Xiang Zhou 0 Huaidong Zhang 0 Xiaofeng Ji 0 Lei Li 0 Lin Huang 0 Linquan Bai 0 Houjin Zhang 0 Eugene A. Permyakov, Russian Academy of Sciences, Institute for Biological Instrumentation, Russian Federation 0 1 Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology , Wuhan, Hubei , China , 2 State Key Laboratory of Microbial Metabolism and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University , Shanghai , China , 3 Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences , Qingdao, Shandong , China 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 C7-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 79phosphate(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. - Funding: This research was financially supported by National Natural Science Foundation of China (No. 31000326 and 31070070), the 973 Program from Ministry of Science & Technology (No. 2009CB118901), and the Ph.D. Training Fund of the Ministry of Education (No. 20100142120002). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Aminocyclitols are a diverse class of bioactive compounds produced by the Streptomyces genus [1]. Based on the chemical structures, aminocylitols can be divided into several groups, such as aminoglycoside, C7N-aminocyclitols and five-membered ring aminocyclitols [2]. The C7N-aminocyclitols, including acarbose, pyralomicin, salbostatin, cetoniacytone A and Val-A, have gained increasing attention due to their extensive applications in agriculture and medicine. Val-A is an antifungal agent isolated from S. hygroscopicus var. limoneus and S. hygroscopicus var. jinggangensis 5008. It is used in many Asian countries as a crop protectant against Rhizoctonia solani, which is a pathogenic fungus responsible for various diseases in plants [3]. The antifungal capability of ValA is attributed to its ability to inhibit trehalase, a trehalosehydrolyzing enzyme. The inhibition of trehalase disrupts the carbon supply and energy production in the fungi, which leads to growth retardation and death of the pathogen [4]. The structure of Val-A consists of an unsaturated valienol unit connected to a saturated validamine unit through a 1-1 a bond, and a glucose unit attached to the validamine moiety [5]. Feeding experiments indicated the 7-carbon unit in the Val-A structure originates from the cyclization of sedoheptulose 7-phosphate [1]. Validamycin biosynthetic gene clusters had been independently cloned from two above-mentioned producers [6,7]. The heterologous expression of eight genes, valABCGKLMN with valL annotated as V7P synthase gene, rendered the surrogate host with validamycin productivity [7]. Through multiple steps, the 2epi-5-epi-valiolone is proposed to be converted to NDP-valienol and validamine 7-phosphate [7,8,9,10,11]. Subsequently, the rare nitrogen-bridged twin-cyclitol core structure in Val-A arises from the condensation of NDP-valienol and validamine 7-phosphate (Figure 1). This coupling reaction was proved to be catalyzed by the pseudoglycosyltransferase ValL, also named as VldE in the biosynthetic gene cluster from S. hygroscopicus var. limon (...truncated)