On the Structural Basis of the Catalytic Mechanism and the Regulation of the Alpha Subunit of Tryptophan Synthase from Salmonella typhimurium and BX1 from Maize, Two Evolutionarily Related Enzymes Victor Kulik 1 , Elisabeth Hartmann 1 , Michael Weyand 2 , Monika Frey 3 Alfons Gierl 3 , Dimitri Niks 4 , Michael F. Dunn 4 and Ilme Schlichting 1 * 1 Max Planck Institut fu ˝r medizinische Forschung Abteilung fu ˝ r Biomolekulare Mechanismen, Jahnstr. 29 69120 Heidelberg, Germany 2 Max Planck Institut fu ˝r Molekulare Physiologie Abeilung fu ˝ r Biophysikalische Chemie, Otto Hahnstr. 11 44227 Dortmund, Germany 3 Lehrstuhl fu ¨r Genetik Technische Universita ¨t Mu ¨nchen, 85 350 Freising Germany 4 Department of Biochemistry University of California Riverside, Riverside, CA 92521 USA Indole is a reaction intermediate in at least two biosynthetic pathways in maize seedlings. In the primary metabolism, the a-subunit (TSA) of the bifunctional tryptophan synthase (TRPS) catalyzes the cleavage of indole 3-glycerol phosphate (IGP) to indole and D-glyceraldehyde 3-phosphate (G3P). Subsequently, indole diffuses through the connecting tunnel to the b-active site where it is condensed with serine to form tryptophan and water. The maize enzyme, BX1, a homolog of TSA, also cleaves IGP to G3P and indole, and the indole is further converted to 2,4-dihydroxy-7- methoxy-2H-1,4-benzoxazin-3(4H)-one, a secondary plant metabolite. BX1 cleaves IGP significantly faster to G3P and indole than does TSA. In line with their different biological functions, these two evolutionary related enzymes differ significantly in their regulatory aspects while catalyzing the same chemistry. Here, the mechanism of IGP cleavage by TSA was analyzed using a novel transition state analogue generated in situ by reaction of 2-aminophenol and G3P. The crystal structure of the complex shows an sp 3 -hybridized atom corresponding to the C3 position of IGP. The catalytic aGlu49 rotates to interact with the sp 3 -hybridized atom and the 3 0 hydroxyl group suggesting that it serves both as proton donor and acceptor in the a-reaction. The second catalytic residue, aAsp60 interacts with the atom corresponding to the indolyl nitrogen, and the catalytically important loop aL6 is in the closed, high activity conformation. Comparison of the TSA and TSA-transition state analogue structures with the crystal structure of BX1 suggests that the faster catalytic rate of BX1 may be due to a stabilization of the active conformation: loop aL6 is closed and the catalytic glutamate is in the active conformation. The latter is caused by a substitution of the residues that stabilize the inactive conformation in TRPS. q 2005 Elsevier Ltd. All rights reserved. Keywords: crystal structure; enzymatic mechanism; transition state ana- logue; structure–function-relationship; TIM barrel *Corresponding author 0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. Present address: M. Weyand, Max Planck Institut fu ˝ r Molekulare Physiologie, Abeilung fu ˝ r Strukturelle Biologie, Otto Hahnstr. 11, 44227 Dortmund, Germany. Abbreviations used: ANS, 8-anilino-1-naphthalenesulfonate; 2AP, 2-aminophenol; GP, D,L-a-glycerol-3-phosphate; G3P, D-glyceraldehyde 3-phosphate; IPP, indole propanol phosphate; TRPS, tryptophan synthase; aL2, loop 2 (aP53- aD60) of the a subunit of tryptophan synthase 10 ; aL6, loop 6 (aR179-aL193) of the a subunit of tryptophan synthase 10 ; TRPS 2amino , tryptophan synthase 2-aminophenol complex; TRPS GP , tryptophan synthase GP complex; TRPS IPP , tryptophan synthase IPP complex. E-mail address of the corresponding author: ilme.schlichting@mpimf-heidelberg.mpg.de doi:10.1016/j.jmb.2005.07.014 J. Mol. Biol. (2005) 352, 608–620