PDBsum entry 1hfb

Lyase

PDB id: 1hfb

Contents

Protein chains

(+ 2 more) 339 a.a. *

Ligands

PEP ×8

Waters ×1251

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Evolution of feedback-Inhibited beta /alpha barrel isoenzymes by gene duplication and a single mutation.

Authors

M.Hartmann, T.R.Schneider, A.Pfeil, G.Heinrich, W.N.Lipscomb, G.H.Braus.

Ref.

Proc Natl Acad Sci U S A, 2003, 100, 862-867. [DOI no: 10.1073/pnas.0337566100]

PubMed id

12540830

Abstract

The betaalpha barrel is the common protein fold of numerous enzymes and was proposed recently to be the result of gene duplication and fusion of an ancient half-barrel. The initial enzyme of shikimate biosynthesis possesses the additional feature of feedback regulation. The crystal structure and kinetic studies on chimera and mutant proteins of yeast 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase from Saccharomyces cerevisiae inhibited by phenylalanine (Aro3p) and DAHP synthase S. cerevisiae inhibited by tyrosine (Aro4p) give insight into important regions for regulation in the enzyme: The loop, which is connecting the two half-barrels, and structural elements added to the barrel are prerequisites for regulation and form a cavity on the N-terminal side of the betaalpha barrel. In the cavity of Aro4p at position 226 is a glycine residue, which is highly conserved in all other tyrosine-regulated DAHP synthases as well. Sequence alignments with phenylalanine-regulated DAHP synthases including Aro3p show a highly conserved serine residue at this position. An exchange of glycine to serine and vice versa leads to a complete change in the regulation pattern. Therefore the evolution of these differently feedback-inhibited isoenzymes required gene duplication and a single mutation within the internal extra element. Numerous additional amino acid substitutions present in the contemporary isoenzymes are irrelevant for regulation and occurred independently.

Figure 2.
Fig. 2. Aro4p structure of S. cerevisiae. (A) Topology plot of DAHP synthase from S. cerevisiae. The -strands and -helices of the central / barrel are shown in orange and yellow, respectively. Loops on the N- and C-terminal sides of the barrel are in cyan and green, respectively. The additional structural elements at the N terminus and between helix 5 and -strand 6 are also shown in cyan. Residues involved in binding of PEP are marked in green. Residues involved in regulation are marked in blue with italic lettering for those identified by random screening and normal lettering for those found by site-directed mutagenesis. Red arrows mark the cutting points in Aro4p for the chimera constructs (Fig. 1A). The dotted 0 strand is part of the second monomer of a dimer and interacts with the 6a and 6b. (B) The crystal structure of DAHP synthase from S. cerevisiae in ribbon presentation. The crystal structures contain a molecule of PEP bound to the active site shown as space-filling models (red and magenta). Secondary structure elements are color-coded as described for A. Shown are views from the C-terminal face of the central barrel (Left), side of the barrel (Center), and N-terminal face of the barrel (Right). The programs MOLSCRIPT 2.1 (21) and RASTER3D (22) were used for the presentation of DAHP synthase.

Figure 5.
Fig. 5. Comparison of the putative effector-binding cavity of the tyrosine- and phenylalanine-regulated DAHP synthases. (A) Amino acid sequence alignment of a part of the putative regulation cavity of the tyrosine- and phenylalanine-regulated DAHP synthases of several organisms. C. albicans, Candida albicans; A. nidulans, Aspergillus nidulans; H. influenzae, Haemophilus influenzae; S. typhimurium, Salmonella typhimurium. (B-D) Accessible surface plots of the tyrosine-regulated DAHP synthase Aro4p from S. cerevisiae (S.c., B and C) and the phenylalanine-inhibited DAHP synthase AroG from E. coli (E.c., D). Surface elements closer than 3 Å to atoms belonging to the N-terminal extension or the inserted sheet are shown in cyan. Residues identified as playing a role in regulation (blue) and for the specificity-related residue (red, G226 in Aro4p and S211 in AroG) are indicated. (B) The orientation is the same as described for Fig. 2B. (C and D) The view is rotated by 40° around the vertical and 30° around the horizontal axis with respect to Fig. 2B. The figure was created with DINO (ref. 23 and www.dino3d.org).