Ouchi Takuya, Tomita Takeo, Miyagawa Tomoharu, Kuzuyama Tomohisa, Nishiyama Makoto
Biotechnology Research Center, The University of Tokyo, Yayoi, Bunkyo-ku, Japan.
Biochem Biophys Res Commun. 2009 Oct 9;388(1):21-7. doi: 10.1016/j.bbrc.2009.07.096. Epub 2009 Jul 24.
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.
为阐明嗜热栖热菌α-氨基己二酸转氨酶（AAA-AT）的底物识别机制，我们测定了与N-(5'-磷酸吡哆醛)-L-谷氨酸（PPE）复合的AAA-AT的晶体结构，分辨率为1.67 Å。晶体结构表明，PPE被与N-(5'-磷酸吡哆醛)-L-α-氨基己二酸（PPA）识别中所见相同的氨基酸残基识别；然而，为了将Glu的γ-羧基固定在一个位置，Glu部分的α碳原子在PPE复合物中向γ-羧基移动了0.80 Å。对Asp活性的显著降低可以通过被Arg23识别的天冬氨酰侧链较短以及结合的Asp的α碳原子的进一步错位来解释。定点诱变表明，Arg23对反应具有双重功能，（i）识别Glu（AAA）的γ（δ）-羧基，（ii）通过改变相互作用伙伴来重排α2螺旋，以便将疏水底物置于合适位置。
