ABSTRACT
LysW has been identified as a carrier protein in the lysine biosynthetic pathway that is active through the conversion of α-aminoadipate (AAA) to lysine. In this study, we found that the hyperthermophilic archaeon, Sulfolobus acidocaldarius, not only biosynthesizes lysine through LysW-mediated protection of AAA but also uses LysW to protect the amino group of glutamate in arginine biosynthesis. In this archaeon, after LysW modification, AAA and glutamate are converted to lysine and ornithine, respectively, by a single set of enzymes with dual functions. The crystal structure of ArgX, the enzyme responsible for modification and protection of the amino moiety of glutamate with LysW, was determined in complex with LysW. Structural comparison and enzymatic characterization using Sulfolobus LysX, Sulfolobus ArgX and Thermus LysX identify the amino acid motif responsible for substrate discrimination between AAA and glutamate. Phylogenetic analysis reveals that gene duplication events at different stages of evolution led to ArgX and LysX.
Subject(s)
Archaeal Proteins/metabolism , Arginine/biosynthesis , Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Lysine/biosynthesis , Sulfolobus acidocaldarius/metabolism , 2-Aminoadipic Acid/metabolism , Archaeal Proteins/chemistry , Archaeal Proteins/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Carrier Proteins/chemistry , Carrier Proteins/genetics , Catalytic Domain , Crystallography, X-Ray , Escherichia coli/genetics , Evolution, Molecular , Gene Duplication , Glutamic Acid/metabolism , Models, Molecular , Ornithine/metabolism , Phylogeny , Protein Binding , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Sulfolobus acidocaldarius/genetics , Thermus/genetics , Thermus/metabolismABSTRACT
To clarify the mechanism for substrate recognition of alpha-aminoadipate aminotransferase (AAA-AT) from Thermus thermophilus, the crystal structure of AAA-AT complexed with N-(5'-phosphopyridoxyl)-l-glutamate (PPE) was determined at 1.67 A resolution. The crystal structure revealed that PPE is recognized by amino acid residues the same as those seen in N-(5'-phosphopyridoxyl)-l-alpha-aminoadipate (PPA) recognition; however, to bind the gamma-carboxyl group of Glu at a fixed position, the Calpha atom of the Glu moiety moves 0.80 A toward the gamma-carboxyl group in the PPE complex. Markedly decreased activity for Asp can be explained by the shortness of the aspartyl side chain to be recognized by Arg23 and further dislocation of the Calpha atom of bound Asp. Site-directed mutagenesis revealed that Arg23 has dual functions for reaction, (i) recognition of gamma (delta)-carboxyl group of Glu (AAA) and (ii) rearrangement of alpha2 helix by changing the interacting partners to place the hydrophobic substrate at the suitable position.
