Biotechnology Letters 25: 909–915, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 909 Functional identification of rub52 gene involved in the biosynthesis of rubradirin Jyoti Maharjan 1 , Kwangkyoung Liou 1 , Hei Chan Lee 1 , Chun-Gyu Kim 2 , Jung-Joon Lee 3 , Jin- Cheol Yoo 4 & Jae Kyung Sohng 1,∗ 1 Institute of Biomolecule Reconstruction, Sun Moon University, #100, Kalsan-ri, Tangjeong-myeon, Asan-si, Chungnam 336-708, South Korea 2 School of Advanced Materials and Process Engineering, Inje University, Kimhae 621-749, Korea 3 Korea Research Institute of Bioscience and Biotechnology, Taejon 305-600, Korea 4 Department of Pharmacy, Chosun University, Kwangju 501-759, Korea ∗ Author for correspondence (Fax: 82-41-544-2919; E-mail: sohng@email.sunmoon.ac.kr) Received 6 March 2003; Revisions requested 14 March 2003; Revisions received 1 April 2003; Accepted 4 April 2003 Key words: deoxygenation, D-rubranitrose, heterologous expression, Streptomyces achromogens Abstract An open reading frame, rub52, has been identified as a gene encoding thymidine diphospho-glucose 2,3- dehydratase by sequence analysis of the rubradirin biosynthetic gene cluster of Streptomyces achromogenes var. rubradiris NRRL3061.The gene codes for a protein consisting of 458 amino acids with calculated molecular mass of 50 862 Da. The gene was amplified and heterologously expressed in Escherichia coli as a soluble His-tagged fusion protein. C-2 deoxygenation functionality of thymidine diphospho-4-keto-6-deoxyglucose was assigned to the rub52 gene product from in vitro enzyme assay. Introduction Secondary metabolites often contain sugar compo- nents that are essential for their biological activities. Many of these unusual sugars are deoxysugars. In general, they all belong to 6-deoxyhexose (6-DOH) These sugars are formed from simple nucleotide- activated carbohydrates (Kirschining et al. 1997). The first two steps leading to the formation of an intermediate thymidine diphospho(TDP)-4-keto-6- deoxyglucose are conserved among all deoxysugar biosynthetic pathways established so far. Besides 6- deoxygenation, C-2 and C-3 deoxygenation occurs in the deoxysugar component of antibiotics. These deoxygenations are followed after C-6 deoxygena- tion. The enzyme catalyzing the C-2 deoxygenation is designated as TDP-glucose 2,3-dehydratase. Several genes encoding a 2,3-dehydratase have been isolated from various antibiotics producing organisms, e.g. eryBVI from Saccharopolyspora erythraea, and dnmT from Streptomyces peucetius (Scotti et al. 1996). The first evidence on the mechanistic activity governed by TDP-glucose 2,3-dehydratase was investigated for the biosynthesis of granaticin deoxysugar moiety at the genetic and biochemical level. TDP-glucose 2,3- dehydratase (Gra ORF27) converts TDP-4-keto-6- deoxyglucose into TDP-3,4-diketo-2,6-deoxyhexose, an unstable intermediate, which undergoes self de- composition into maltol and thymidine diphosphate (TDP) (Draeger et al. 1999). To trace the biosynthetic pathway of 2,3,6-tri- deoxy-3-C-methyl-4-O-methyl-3-nitro-D-xylohexose, D-rubranitrose, as a sole sugar moiety of antibi- otic rubradirin (6) (Figure 1), we have cloned the rubradirin biosynthetic gene cluster. Based on avail- able knowledge relating to the biosynthesis of 2,6- deoxysugars, the biosynthesis of the rubranitrose would follow the pathway via an intermediate, TDP- 3,4-diketo-2,6-deoxyhexose (4) (Figure 1). The first two steps are conserved, and are catalyzed by TDP- glucose synthase (Rub53) and TDP-glucose 4,6-