PDBsum entry 1fuy

Lyase

PDB id: 1fuy

Contents

Protein chains

262 a.a. *

393 a.a. *

Ligands

FIP

PLP

Metals

_NA

Waters ×190

* Residue conservation analysis

PDB id:

1fuy

Name:

Lyase

Title:

Crystal structure of betaa169l/betac170w double mutant of tryptophan synthase complexed with 5-fluoro-indole-propanol phosphate

Structure:

Tryptophan synthase alpha chain. Chain: a. Engineered: yes. Tryptophan synthase beta chain. Chain: b. Engineered: yes. Mutation: yes

Source:

Salmonella typhimurium. Organism_taxid: 602. Cell_line: cb149. Gene: trpa/trpb. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Tetramer (from PDB file)

Resolution:

2.25Å R-factor: 0.178 R-free: 0.224

Authors:

M.Weyand,I.Schlichting

Key ref:

M.Weyand and I.Schlichting (2000). Structural basis for the impaired channeling and allosteric inter-subunit communication in the beta A169L/beta C170W mutant of tryptophan synthase. J Biol Chem, 275, 41058-41063. PubMed id: 11034989 DOI: 10.1074/jbc.C000479200

Date:

18-Sep-00 Release date: 17-Jan-01

Protein chain

P00929  (TRPA_SALTY) -  Tryptophan synthase alpha chain from Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)

Seq: 268 a.a.

Struc: 262 a.a.

Protein chain

P0A2K1  (TRPB_SALTY) -  Tryptophan synthase beta chain from Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)

Seq: 397 a.a.

Struc: 393 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chains A, B: E.C.4.2.1.20 - tryptophan synthase.
• Pathway: Tryptophan Biosynthesis
• Reaction: (1S,2R)-1-C-(indol-3-yl)glycerol 3-phosphate + L-serine = D-glyceraldehyde 3-phosphate + L-tryptophan + H2O

(1S,2R)-1-C-(indol-3-yl)glycerol 3-phosphate + L-serine (Bound ligand (Het Group name = FIP) matches with 85.00% similarity) = D-glyceraldehyde 3-phosphate + L-tryptophan + H2O

• Cofactor: Pyridoxal 5'-phosphate

Pyridoxal 5'-phosphate (Bound ligand (Het Group name = PLP) matches with 93.75% similarity)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.C000479200

J Biol Chem 275:41058-41063 (2000)

PubMed id: 11034989

Structural basis for the impaired channeling and allosteric inter-subunit communication in the beta A169L/beta C170W mutant of tryptophan synthase.

M.Weyand, I.Schlichting.

ABSTRACT

We determined the 2.25 A resolution crystal structure of the betaA169L/betaC170W mutant form of the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium complexed with the alpha-active site substrate analogue 5-fluoro-indole-propanol-phosphate to identify the structural basis for the changed kinetic properties of the mutant (Anderson, K. S., Kim, A. Y., Quillen, J. M., Sayers, E., Yang, X. J., and Miles, E. W. (1995) J. Biol. Chem. 270, 29936-29944). Comparison with the wild-type enzyme showed that the betaTrp(170) side chain occludes the tunnel connecting the alpha- and beta-active sites, explaining the accumulation of the intermediate indole during a single enzyme turnover. To prevent a steric clash between betaLeu(169) and betaGly(135), located in the beta-sheet of the COMM (communication) domain (betaGly(102)-betaGly(189)), the latter reorganizes. The changed COMM domain conformation results in a loss of the hydrogen bonding networks between the alpha- and beta-active sites, explaining the poor activation of the alpha-reaction upon formation of the aminoacrylate complex at the beta-active site. The 100-fold reduced affinity for serine seems to result from a movement of betaAsp(305) away from the beta-active site so that it cannot interact with the hydroxyl group of a pyridoxal phosphate-bound serine. The proposed structural dissection of the effects of each single mutation in the betaA169L/betaC170W mutant would explain the very different kinetics of this mutant and betaC170F.

Selected figure(s)

Figure 1.
Fig. 1. A, residual B-factor plot for the wild-type TRPSIPP (green) and the A169L/ C170WF-IPP (black) structures (top). The plot of the residual B-factor ratio (mutant B[fac]/wild-type B[fac]) is shown. Although both complexes have different mean B-factors, the B-factor comparison allows the identification of changes within the COMM domain (bottom). B, C[ ]r.m.s. deviation (RMSD) of TRPSIPP and A169L/ C179WF-IPP for the (top) and (bottom) subunits. Points I[ ]and I[ ]represent flexible surface residues. The insert shows a detailed view of the r.m.s. deviation and the secondary structure assignment within the COMM domain.

Figure 2.
Fig. 2. A, stereo drawing of Sigma A-weighted 2 mF[o] DF[c] maps (20) contoured at 1 around the A169L/ C170W mutation site showing the good definition of the two new side chains and the reorientation of both gating residues tyrosine Tyr279 and phenylalanine Phe^280. The figure was prepared using BOBSCRIPT (24) and RASTER3D (25, 26). B, stereo view of the superposition of the COMM domains of wild-type TRPSIPP and A169L/ C170WF-IPP. Mutated and gating residues of the -subunit are shown in a ball-and-stick representation. C trace and residues of wild-type TRPSIPP are red ( -subunit), dark blue, ( -subunit), and yellow (COMM domain, the double mutant is green, carbon atoms of the -ligands and the cofactor PLP are gray, oxygen atoms red, nitrogen atoms blue, and phosphate is magenta. Panels A and B are related by an ~90° rotation around the axis perpendicular to the paper plane. The figure was prepared using MOLSCRIPT (27) and RASTER3D (25, 26).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2000, 275, 41058-41063) copyright 2000.

Author's comment

We determined the 2.25 Å resolution crystal structure of the betaA169L/betaC170W mutant form of the tryptophan synthase alapha2beta2 complex from Salmonella typhimurium complexed with the alpha-active site substrate analogue 5-fluoro-indole-propanol-phosphate to identify the structural basis for the changed kinetic properties of the mutant (Anderson, K. S., Kim, A. Y., Quillen, J. M., Sayers, E., Yang, X. J., and Miles, E. W. (1995) J. Biol. Chem. 270, 29936–29944). Comparison with the wild-type enzyme showed that the betaTrp170 side chain occludes the tunnel connecting the alpha- and beta-active sites, explaining the accumulation of the intermediate indole during a single enzyme turnover. To prevent a steric clash between betaLeu169 and betaGly135, located in the beta-sheet of the COMM (communication) domain (betaGly102-betaGly189), the latter reorganizes. The changed COMM domain conformation results in a loss of the hydrogen bonding networks between the alpha- and beta-active sites, explaining the poor activation of the alpha-reaction upon formation of the aminoacrylate complex at the beta-active site. The 100-fold reduced affinity for serine seems to result from a movement of betaAsp305 away from the beta-active site so that it cannot interact with the hydroxyl group of a pyridoxal phosphate-bound serine. The proposed structural dissection of the effects of each single mutation in the betaA169L/betaC170W mutant would explain the very different kinetics of this mutant and betaC170F.