STRUCTURE NOTE Crystal Structure of the Escherichia Coli Tas Protein, an NADP(H)-Dependent Aldo-Keto Reductase Galina Obmolova, 1 Alexey Teplyakov, 1 Pavel P. Khil, 2 Andrew J. Howard, 3 R. Daniel Camerini-Otero, 2 and Gary L. Gilliland 1 * 1 Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute and the National Institute of Standards and Technology, Rockville, Maryland 2 Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 3 Center for Synchrotron Radiation Research and Instrumentation, Biological, Chemical and Physical Sciences Department, Illinois Institute of Technology, Chicago, Illinois Introduction. The Tas gene of Escherichia coli encodes an uncharacterized protein of 38.5-kDa molecular weight. The amino acid sequence shows homology to the family of aldo-keto reductases that reduce aldehyde or ketone func- tional groups to primary or secondary alcohols.The basic chemical reaction is the stereospecific transfer of hydride between the nicotinamide ring of NADPH a and a carbon center on the substrate molecule. The aldo-keto reductases metabolize a wide range of small molecule substrates including sugars, aliphatic aldehydes, aromatic hydrocar- bons, steroids, and prostaglandins. 1 For many members of the family, however, the physiological substrates are not known. Crystallographic studies of human aldose reductase 2–4 have established the molecular architecture, geometry of cofactor binding, and the catalytic mechanism of aldo-keto reductases. All members of the family have a (/) 8 TIM barrel fold. The catalytic site with the bound NADP(H) cofactor is located at the C-terminal edge of the -strands. Loops surrounding the active site form the substrate recognition cylindrical surface. Crystal complexes with various inhibitors 5–8 have indicated the ability of aldo- keto reductases to accommodate a wide range of sub- strates due to the conformational flexibility of the recogni- tion loops. Tas was identified as the gene able to complement the tyrosine requirement in E. coli tyrosine auxotroph strains under starvation conditions, 9 hence the name Tas (ty- rosine auxotrophy suppressor). Most likely, the enzymatic activity of the Tas protein directly substitutes for the lacking prephenate dehydrogenase activity of the auxo- troph strain; however, the involvement of Tas in the mutation process resulting in slow-growing Tyr + rever- tants cannot be ruled out. Tas was among the genes significantly induced in response to the DNA damage caused by mitomycin C 10 and therefore may be part of a general protective response. The crystal structure determination of Tas was under- taken as part of a structural genomics effort 11 (http:// s2f.carb.nist.gov) to assist with the functional assignment of the protein. The Tas protein from E. coli was cloned, expressed, and the crystal structure determined at 1.6-Å resolution. Materials and Methods. Cloning, expression, and purifi- cation. The Tas gene was polymerase chain reaction (PCR) amplified from E. coli MG1655 genomic DNA and sub- cloned into a pDONR201 plasmid using the Gateway technology (Invitrogen). For expression, the coding se- quence was transferred into a pDEST14 plasmid using site-specific recombination (Invitrogen). The protein was produced in E. coli strain BL21 Star (DE3) (Invitrogen) that was transformed with pDEST14. Cells were grown on LB media containing 100 g/L ampicillin at 37°C to an A 600 of 0.6 and induced with 1 mM isopropyl -D- thiogalactoside for 3 h. The protein was purified by column chromatography in two steps using Source 30Q (Pharma- cia) and Butyl-560M (Toyopearl). Abbreviations: NADPH, nicotinamide adenine dinucleotide phos- phate; TIM, triosephosphate isomerase; KVB2, voltage-dependent K + channel  subunit ; RMSD, root mean square deviation; PCR, polymer- ase chain reaction; IMCA-CAT, Industrial Macromolecular Crystallog- raphy Collaborative Access Team; APS, Advanced Photon Source; CCD, charge-coupled device. Grant sponsor: National Institutes of Health; Grant number: P01- GM57890. Certain commercial materials, instruments, and equipment are identified in this article to specify the experimental procedure as completely as possible. In no case does such identification imply a recommendation or endorsement by the National Institute of Stan- dards and Technology nor does it imply that the materials, instru- ments, or equipment identified is necessarily the best available for the purpose. The accepted SI units of concentration, mol/L, and of unified atomic mass unit, u, have been represented by the symbols M and Da, respectively, to conform to the conventions of this journal. *Correspondence to: Gary Gilliland, Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, and the National Institute of Standards and Technology, 9600 Gudelsky Drive, Rockville, MD 20850; E-mail: gary.gilliland@nist.gov Received 21 November 2002; Accepted 4 December 2002 PROTEINS: Structure, Function, and Genetics 53:323–325 (2003) © 2003 WILEY-LISS, INC.