1. Jun Xu, Taifo Mahmud* and Heinz G. Floss* Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195 Identification and Characterization of 27-O-Demethylrifamycin SV Methyltransferase Background Rifamycin B, one of the prominent members of the ansamitocin family, is a microbial secondary metabolite antibiotic produced by Amicolatopsis mediterranei. Its synthetically modified derivatives are clinically used in the treatment of tuberculosis, leprosy, and AIDS-related mycobacterial infections. Recently, rifamycin derivatives have also shown excellent activity against HIV and oncogenic viruses. However, many pathogen bacteria develop resistance to rifamycins with high frequency. The ansa chain of Rifamycin B has been proposed to be synthesized from acetate and propionate units catalyzed by a large multifunctional complex enzyme, type I polyketide synthases (PKSs). The starter molecule for this polyketide assembly is derived from 3-amino-5-hydroxybenzoic acid (AHBA). Although great progress has been made in the last few years on the understanding of the biosyntheses of AHBA and the ansa chain of rifamycin B, little is known regarding the post-PKS modification genes and the tailoring processes leading to rifamycin B. Here we report the identification and characterization of the Orf14, an S-adenosyl-L-methionine dependent methyltransferase that is involved in post-PKS modification of rifamycin B biosynthesis. Cloning and Inactivation of orf 14 Isolation and Structure Elucidation of Accumulated Compound Orf14 was subcloned into Bluescript II KS -, and frame shift inactivation was done at XhoI site. Double homologous recombination mutant of Amycolatopsis mediterranei S699 (mutant MT1401XH) was obtained. Oxidation of 27-O-Demethylrifamycin SV to 27-O-Demethylsifamycin S Catalyzed by Divalent Ions Genetic Organization of the Rifamycin Biosynthetic Gene Cluster From fermentation broth of mutant MT1401XH, 27-O-demethylrifamycin SV was isolated as a major metabolite, together with traces of 25-O- deacetyl-27-O-demethylrifamycin SV, 27-O-demethylrifamycin S, and rifamycin W. Their chemical structures were elucidated based on mass spectroscopy and NMR data. Over Expression of orf 14 In order to obtain His 6 -fusion protein, two primers were designed and PCR was carried out to amplify the orf14. The PCR product was inserted into pRSET B, a T7 promoter expression vector, and the product was transferred into E. coli BL21 by heat-pulse transformation. SDS polyacrylamide gel electrophoresis confirmed that the right size protein was over-expressed. The protein was purified by Ni- NTA spin column. Characterization of Orf14 D. Substrate Specificity E. Kinetic parameters Conclusion Proposed biosynthetic pathway to Rifamycin B. 27-O-Demethylrifamycin SV 27-O-Demethylrifamycin S Cu 2+ Mn 2+ Ca 2+, Co 2+, Fe 2+, Mg 2+, Ni 2+, Zn 2+ Acknowledgements This work was supported by a research grant AI 20264 from the National Institutes of Health. Acquisition of the Bruker Esquire ion trap mass spectrometer was supported by the National Science Foundation through grant No. 9807748. C. Effects of Divalent ions 1 4 2 ESI-MS data for enzyme assays with different rifamycins as substrate: 1. 27-O-Demethylrifamycin SV; 2. 27-O-Demethylrifamycin S; 3. Rifamycin W; 4. 25-O-Deacyl-27-O- demethylrifamycin SV 27-O-Demethylrifamycin SV appears to be the right substrate for the methyltransferase, Orf14 (Figure 1) 38 KD Vmax=142.8 nM/sec Km=19.3 uM 3 Rif SV A. Optimal BufferB. Optimal pH Value