proteins STRUCTURE O FUNCTION O BIOINFORMATICS Potential anti-bacterial drug target: Structural characterization of 3,4-dihydroxy- 2-butanone-4-phosphate synthase from Salmonella typhimurium LT2 Pankaj Kumar, Mirage Singh, Ruchi Gautam, and Subramanian Karthikeyan * Institute of Microbial Technology, Council of Scientific and Industrial Research (CSIR), Sector 39-A, Chandigarh 160 036, India INTRODUCTION The Gram negative bacteria Salmonella enterica serovar Typh- imurium (Salmonella typhimurium) causes gastroenteritis some- times referred to as salmonellosis, in human and animals. 1,2 This bacterium is gaining resistance to the commonly used anti- biotics like fluoroquinolone and third-generation of cephalospo- rin 3 resulting in a severe limitation on the possibilities of effec- tive treatment of human and animal infections. Therefore, iden- tification of novel drug targets is urgently needed to combat this multidrug resistant pathogen. Recently, it has been shown that the Gram negative bacteria like Escherichia coli and Salmo- nella sp. are absolutely dependent on the endogenous biosynthe- sis of riboflavin for their growth as they are devoid of any uptake system for flavins. 4 Moreover, riboflavin biosynthesis pathway is absent in humans and they obtain riboflavin from their diet. Therefore, the enzymes involved in riboflavin biosyn- thesis are considered as potential anti-bacterial drug targets as this pathway is essential for the pathogens but absent in humans. The three-dimensional structure determination of enzymes involved in riboflavin biosynthesis pathway will help us to understand its structure-function relationship and provides a platform to identify new anti-bacterial drug molecules by rational structure-based drug designing. 3,4-Dihydroxy-2-butanone-4-phosphate synthase (DHBPS) is one of the first enzymes of the riboflavin biosynthesis pathway that catalyzes the conversion of ribulose-5-phosphate (Ru5P) to 3,4-dihydroxy-2-butanone-4-phosphate (DHBP) and formate (Fig. 1). 5 From another branch of riboflavin biosynthesis pathway, four enzymes [guanosine triphosphate (GTP) cyclohydrolase II, pyrimi- dine deaminase, pyrimidine reductase, and a hitherto unknown enzyme] catalyzes the formation of 4-ribitylamino-5-amino-2,6- dihydroxypyrimidine (RAADP) from GTP. 6–10 Lumazine synthase Grant sponsor: CSIR; Grant number: NWP05; Grant sponsor: Department of Biotechnol- ogy, Government of India *Correspondence to: Dr. Subramanian Karthikeyan, Institute of Microbial Technology (CSIR), Sector 39-A, Chandigarh 160 036, India. E-mail: skarthik@imtech.res.in (or) skarthik14@gmail.com. Received 9 April 2010; Revised 28 June 2010; Accepted 14 July 2010 Published online 30 July 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/prot.22837 ABSTRACT 3,4-Dihydroxy-2-butanone-4-phosphate synthase (DHBPS) encoded by ribB gene is one of the first enzymes in riboflavin biosynthesis pathway and cata- lyzes the conversion of ribulose-5-phosphate (Ru5P) to 3,4-dihydroxy-2-butanone-4-phosphate and for- mate. DHBPS is an attractive target for developing anti-bacterial drugs as this enzyme is essential for pathogens, but absent in humans. The recombinant DHBPS enzyme of Salmonella requires magnesium ion for its activity and catalyzes the formation of 3,4- dihydroxy-2-butanone-4-phosphate from Ru5P at a rate of 199 nmol min 21 mg 21 with K m value of 116 lM at 378C. Further, we have determined the crystal structures of Salmonella DHBPS in complex with sul- fate, Ru5P and sulfate-zinc ion at a resolution of 2.80, 2.52, and 1.86 A ˚ , respectively. Analysis of these crystal structures reveals that the acidic loop (residues 34–39) responsible for the acid-base catalysis is disordered in the absence of substrate or metal ion at the active site. Upon binding either substrate or sulfate and metal ions, the acidic loop becomes stabilized, adopts a closed conformation and interacts with the sub- strate. Our structure for the first time reveals that binding of substrate Ru5P alone is sufficient for the stabilization of the acidic active site loop into a closed conformation. In addition, the Glu38 residue from the acidic active site loop undergoes a conformational change upon Ru5P binding, which helps in position- ing the second metal ion that stabilizes the Ru5P and the reaction intermediates. This is the first structural report of DHBPS in complex with either substrate or metal ion from any eubacteria. Proteins 2010; 78:3292–3303. V V C 2010 Wiley-Liss, Inc. Key words: DHBPS; ribulose-5-phosphate; crystal struc- ture; riboflavin biosynthesis; antimicrobial target; flavin mono nucleotide; flavin adenine dinucleotide. 3292 PROTEINS V V C 2010 WILEY-LISS, INC.