Crystal structure of the yeast His6 enzyme suggests a reaction mechanism

Abstract

The Saccharomyces cerevisiae His6 gene codes for the enzyme phosphoribosyl-5-amino-1-phosphoribosyl-4-imidazolecarboxamide isomerase, catalyzing the fourth step in histidine biosynthesis. To get an insight into the structure and function of this enzyme, we determined its X-ray structure at a resolution of 1.30 A using the anomalous diffraction signal of the protein's sulphur atoms at 1.77 A wavelength. His6 folds in an (alpha/beta)8 barrel similar to HisA, which performs the same function in bacteria and archaea. We found a citrate molecule from the buffer bound in a pocket near the expected position of the active site and used it to model the open form of the substrate (phosphoribulosyl moiety), which is a reaction intermediate. This model enables us to identify catalytic residues and to propose a reaction mechanism where two aspartates act as acid/base catalysts: Asp134 as a proton donor for ring opening, and Asp9 as a proton acceptor and donor during enolization of the aminoaldose. Asp9 is conserved in yeast His6 and bacterial or archaeal HisA sequences, and Asp134 has equivalents in both HisA and TrpF, but they occur at a different position in the protein sequence.

Figure 1.

(A) Reaction catalyzed by His6: ProFAR (N-(5′-phospho-D-ribosylformimino)-5-amino-1-(5″-phosphoribosyl)-4-imidazolecarboxamide) ⇆ PRFAR (N-(5′-phospho-D-1′-ribulosylformimino)-5-amino-1-(5″-phosphoribosyl)-4-imidazolecarboxamide. (B) Proposed reaction mechanism of His6. The substrate binds as the closed form and ring opening is catalyzed by protonation mediated by Asp134 yielding the Shiff's base. After opening of the ring, the substrate is repositioned in front of the catalytic Asp9. This aspartate abstracts a proton from the C2′carbon and transfers it to the C1′position yielding the ribulose product.

Figure 2.

(A) Ribbon representation of His6 colored from blue (N_ter) to red (C_ter) with the bound citrate molecule shown as sticks. (B) View of citrate modeled into the residual F_o−F_c electron density map contoured at 3.5σ. Hydrogen bonds between citrate, ethylene glycol, and His6 are depicted by red dashed lines. (C) Stereo view representation of the active sites of Tm HisF (magenta; PDB code 1GPW) and the S. cerevisiae His6 (yellow). For clarity, only the active site is shown. The phosphate ion (phosphorus atom in green) bound to the HisF enzyme is shown as sticks. The citrate molecule bound to yeast His6 and a model for the D-ribulose-5-phosphate moiety of the His6 product, are shown in sticks (carbon atoms in yellow) and ball-and-sticks (carbon atoms, blue; phosphorus atom, magenta), respectively. The nitrogen position of the N-(5′-phospho-D-ribosylformimino) is indicated by capital N. (D) Stereo presentation of the active site of TrpF bound to the reaction product.