DOI: 10.1002/cbic.201200173 Analysis of the Mildiomycin Biosynthesis Gene Cluster in Streptoverticillum remofaciens ZJU5119 and a Characterization of MilC, Hydroxymethylcytosylglucuronic Acid Synthase Jun Wu, [a] Li Li, [a, b] Zixin Deng, [a] T. Mark Zabriskie, [c] and Xinyi He* [a] Introduction Powdery mildews, which are caused by the mildew fungus, are common diseases on ornamental plants in the urban land- scape. Because mildew is seriously pernicious to many different plant species, it is considered to be a highly important disease in agriculture. In Europe, it is one of the most consistently damaging diseases of grain cereals. [1] Mildiomycin (MIL, Scheme 1), a peptidyl-nucleoside antibiotic produced by Strep- toverticillum remofaciens, can strongly inhibit the powdery mildew disease on plants. [2] The mode of action of mildiomy- cin, unlike those of the uridine-based nikkomycins [3, 4] and poly- oxins, [5, 6] which target cellulose biosynthesis in fungi, is to block the peptidyl-transferase center of the larger ribosomal subunit. [7] It is less active in the inhibition of RNA or DNA syn- thesis of mammalian cells and plants and is therefore widely used in agriculture and horticulture in Japan. Although the peptidyl nucleoside family of antibiotics en- compasses a structurally diverse group of compounds and many of them exhibit potent and varied biological activities, [8] the structure of MIL is similar to those of many cytosine deriva- tive nucleoside antibiotics, such as gougerotin, bagouger- amine B, arginomycin (ARG), and blasticidin S (BLS). Moreover, a core moiety, 4-aminoacyl-4-deoxyhexopyranose uronic acid cytosine, is shared by MIL, BLS, and ARG (Scheme 1, bold part). This uncommon structure is different from puromycin, the nu- cleoside peptidyl part of which is directly derived from primary metabolism. The earliest committed intermediate in the bio- synthesis of BLS is cytosylglucuronic acid (CGA), formed by coupling of UDP-glucuronic acid and cytosine, catalyzed by a CGA synthase. [9, 10] BLS is the only hexose-based peptidyl nucleoside that has had its biosynthesis gene cluster reported. [11] In BLS formation, a novel CMP hydrolase, BlsM, was heterologously expressed and demonstrated to convert CMP into free cytosine, thus ini- tiating the biosynthetic process. [12] Because of the structural similarity between BLS and MIL, the coding sequence of blsM was used to clone a counterpart, milB, from the MIL producer. In our previous report, the functions of milA and milB were elu- cidated. [13] MilA was shown to catalyze the hydroxymethylation Mildiomycin (MIL) is a peptidyl-nucleoside antibiotic produced by Streptoverticillum remofaciens ZJU5119 that exhibits strong inhibitory activity against powdery mildew. The entire MIL bio- synthesis gene cluster was cloned and expressed in Streptomy- ces lividans 1326. Systematic gene disruptions narrowed down the cluster to 16 functional ORFs and identified the boundaries of the gene cluster. A putative cytosylglucuronic acid (CGA) synthase gene, milC, was disrupted in Sv. remofaciens and het- erologously expressed in E. coli. An in vitro assay revealed that purified MilC could utilize either cytosine or hydroxymethyl- cytosine as substrate to yield CGA or hydroxymethyl-CGA (HM- CGA), respectively. MilG is believed to be a key enzyme in the MIL biosynthesis pathway and contains the C XXX C XX C motif characteristic of members of the radical S-adenosyl methionine (SAM) superfamily. Disruption of milG leads to accumulation of HM-CGA. Labeling experiments with 13 C 6 -l-arginine indicated that decarboxylation at C5 of the pyranoside ring was coupled with the attachment of 5-guanidino-2,4-dihydroxyvalerate side chain through C ÀC bond formation. In contrast, exogenous 13 C 6 -labeled 4-hydroxy-l-arginine was not incorporated into the MIL structure. Comparative analysis of the 16 MIL ORFs with counterparts involved in the biosynthesis of the structur- ally similar compound blasticidin S, along with the results above, provide insight into the complete MIL biosynthetic pathway. [a] J. Wu, + Dr. L. Li, + Prof. Z. Deng, Dr. X. He State Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology Shanghai Jiao Tong University 1954 Huashan Road, Shanghai, 200030 (China) E-mail : xyhe@sjtu.edu.cn [b] Dr. L. Li + Engineering Research Center of Industrial Microbiology (Ministry of Education), and College of Life Sciences, Fujian Normal University Fuzhou, Fujian 350108 (China) [c] Prof. T. M. Zabriskie Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University Corvallis, OR 97331-3507 (USA) [ + ] These authors contribute equally to this work. Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201200173. ChemBioChem 0000, 00, 1 – 10  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim &1& These are not the final page numbers! ÞÞ