The Structure Biology Research On AnPRT(Anthranilate Phosphoribosyltransferase)from Saccharomyces Cerevisiae

Tryptophan is not only an essential component for protein synthesis in organisms,but also regulates synthesis of many proteins.Moreover,the abnormality of tryptophan biosynthesis will have a significant impact on the normal life activities of organisms.In Saccharomyces cerevisiae,anthranilate phosphoribosyltransferase catalyzes the second step of tryptophan biosynthesis by transferring the phosphoribose group of 5’phosphoribosyl-1’-pyrophosphate(PRPP)to the amino group of anthranilate(ANT),which generates phosphoribosylanthranilate(PRA)constituting the basic backbone of tryptophan.In order to study the substrate binding mode of the Sc-AnPRT protein,we solve the crystal structure of Sc-AnPRT protein in isolation,in complex with substrates and with substrate analogs.Comparing Sc-AnPRT with its homology protein,we discovered that the main structural difference is the length of the N-terminal α helix.The substrate PRPP was bound to Sc-AnPRT by making hydrogen bonds with serval residues from the conserved motifs GTGGD,NXST and KHGX.The anthranilate analogue 4FA was bound to Sc-AnPRT by forming hydrogen bond with a conserved arginine R197,and these interactions were strengthened by the hydrogen bond network of water molecules.Comparing the above structures,we discovered that the substrates can bind independently to the protein.Two variants were designed for investigating the role of the S121and G141 in substrate binding:Sc-AnPRTS121A and Sc-AnPRTG141N.In the structure of variant Sc-S 121A in complex with PRPP·Mg2+,only magnesium ions was observed in the active site.Combining the previous structural information,we speculated PRPP binding mode is affected for the variant of S121 A,which can’t form magnesium ion binding site.The anthranilate binding mode of Sc-AnPRTG141N associating with the anthranilate analogue 4FA was consistent with that observed in the wild-type protein,indicating that residue of G141 isn’t crucial for anthranilate binding in Sc-AnPRT.In addition,the combination of multiple sequence alignment with overall structure alignment suggested that Sc-AnPRT may have two anthranilate binding sites.Taken together,we speculated that the Sc-AnPRT may have a novel mechanism that neither needing to regulate the sequence of substrate binding by conformational rearrangement of flexible loops,nor rotate intermolecular domain to initiate enzymatic reaction.