Dual roles of a conserved pair, Arg23 and Ser20, in recognition of multiple substrates in alpha-aminoadipate aminotransferase from Thermus thermophilus.
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.
Ouchi
Takuya
T
Biotechnology Research Center, The University of Tokyo, Yayoi, Bunkyo-ku, Japan.
Tomita
Takeo
T
Miyagawa
Tomoharu
T
Kuzuyama
Tomohisa
T
Nishiyama
Makoto
M
D051307:2-Aminoadipate Transaminase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015074:2-Aminoadipic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D005971:Glutamates / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D011725:Pyridines / Q000737:chemistry / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology*
D005971:Glutamates; C430143:N-(5'-phosphopyridoxyl)-glutamate; C540866:N-phosphopyridoxyl-alpha-aminoadipate; D011725:Pyridines; D015074:2-Aminoadipic Acid; D012694:Serine; D011732:Pyridoxal Phosphate; D001120:Arginine; D051307:2-Aminoadipate Transaminase