Mechanism for multiple-substrates recognition of alpha-aminoadipate aminotransferase from Thermus thermophilus.
Alpha-aminoadipate aminotransferase (AAA-AT), a homolog of mammalian kynurenine aminotransferase II (Kat II), transfers an amino group to 2-oxoadipate to yield alpha-aminoadipate in lysine biosynthesis through the alpha-aminoadipate pathway in Thermus thermophilus. AAA-AT catalyzes transamination against various substrates, including AAA, glutamate, leucine, and aromatic amino acids. To elucidate the structural change for recognition of various substrates, we determined crystal structures of AAA-AT in four forms: with pyridoxal 5'-phosphate (PLP) (PLP complex), with PLP and leucine (PLP/Leu complex), with N-phosphopyridoxyl-leucine (PPL) (PPL complex), and with N-phosphopyridoxyl-alpha-aminoadipate (PPA) at 2.67, 2.26, 1.75, and 1.67 A resolution, respectively. The PLP complex is in an open state, whereas PLP/Leu, PPL, and PPA complexes are in closed states with maximal displacement (over 7 A) of the alpha2 helix and the beta1 strand in the small domain to cover the active site, indicating that conformational change is induced by substrate binding. In PPL and PLP/Leu complexes, several hydrophobic residues on the alpha2 helix recognize the hydrophobic side chain of the bound leucine moiety whereas, in the PPA complex, the alpha2 helix rotates to place the guanidium moiety of Arg23 on the helix at the appropriate position to interact with the carboxyl side chain of the AAA moiety. These results indicate that AAA-AT can recognize various kinds of substrates using the mobile alpha2 helix. The crystal structures and site-directed mutagenesis revealed that intersubunit-electrostatic interactions contribute to the elevated thermostability of this enzyme.
Tomita
Takeo
T
Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Miyagawa
Tomoharu
T
Miyazaki
Takashi
T
Fushinobu
Shinya
S
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; D004795:Enzyme Stability; D006358:Hot Temperature; D006801:Humans; D007930:Leucine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology*; D000637:Transaminases / Q000737:chemistry / Q000378:metabolism
C540866:N-phosphopyridoxyl-alpha-aminoadipate; C540865:N-phosphopyridoxylleucine; D015074:2-Aminoadipic Acid; D011732:Pyridoxal Phosphate; D000637:Transaminases; D051307:2-Aminoadipate Transaminase; C021481:kynurenine-oxoglutarate transaminase; D007930:Leucine