TY - JOUR
T1 - 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
AU - Kulik, Victor
AU - Hartmann, Elisabeth
AU - Weyand, Michael
AU - Frey, Monika
AU - Gierl, Alfons
AU - Niks, Dimitri
AU - Dunn, Michael F.
AU - Schlichting, Ilme
N1 - Funding Information:
We thank Georg Holtermann for help and excellent technical assistance, Herbert Zimmermann for the nomenclature of the transition state analogue, the staff at the beamlines at the ESRF, Grenoble, France for support during data collection, Roger S. Goody for continuous encouragement, support, and hospitality, and the DFG (I.S.) for funding. The research also was supported by the National Institutes of Health (USA) grant RO1 GM55749 to M.F.D.
PY - 2005/9/23
Y1 - 2005/9/23
N2 - Indole is a reaction intermediate in at least two biosynthetic pathways in maize seedlings. In the primary metabolism, the α-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 β-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 sp3-hybridized atom corresponding to the C3 position of IGP. The catalytic αGlu49 rotates to interact with the sp3-hybridized atom and the 3′ hydroxyl group suggesting that it serves both as proton donor and acceptor in the α-reaction. The second catalytic residue, αAsp60 interacts with the atom corresponding to the indolyl nitrogen, and the catalytically important loop αL6 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 αL6 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.
AB - Indole is a reaction intermediate in at least two biosynthetic pathways in maize seedlings. In the primary metabolism, the α-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 β-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 sp3-hybridized atom corresponding to the C3 position of IGP. The catalytic αGlu49 rotates to interact with the sp3-hybridized atom and the 3′ hydroxyl group suggesting that it serves both as proton donor and acceptor in the α-reaction. The second catalytic residue, αAsp60 interacts with the atom corresponding to the indolyl nitrogen, and the catalytically important loop αL6 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 αL6 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.
KW - Crystal structure
KW - Enzymatic mechanism
KW - Structure-function-relationship
KW - TIM barrel
KW - Transition state analogue
UR - https://www.scopus.com/pages/publications/24044547672
U2 - 10.1016/j.jmb.2005.07.014
DO - 10.1016/j.jmb.2005.07.014
M3 - Article
C2 - 16120446
AN - SCOPUS:24044547672
SN - 0022-2836
VL - 352
SP - 608
EP - 620
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 3
ER -