Structure-function relationship of novel lysine biosynthetic enzymes and analysis of molecular evolution of the pathway

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1)Detailed analysis of homoaconitase from Thermus thermophilus revealed that the enzyme catalyzes the 3rd reaction but could not the 2nd reaction of the pathway. 2)X-ray structures of α-aminoadipate aminotransferase (AAA-AT) were determined in the several forms. AAA-AT recognizes not only 2-oxoisocaproate, an intermediate in lysine biosynthesis, but also the corresponding compounds in the leucine and phenylalanine biosyntheses. The X-ray structure indicates that the mobile at helix located N-terminus is responsible for the broad substrate specificity of this enzyme, and that an arginine residue on the helix plays a crucial role in the catalytic function. 3)By DNA shuffling technique, homoisocitrate dehydrogenase was successfully converted to the enzyme that utilizes 2-isopropylmalate that is the corresponding compound in leucine biosynthesis but is not recognized as a substrate by wild-type homoisocitrate dehydrogenase. Analysis of the resulting evolved enzyme and effect of each amino acid substitution revealed that all the replacements introduced in the evolved enzyme had positive effect on the change in the substrate specificity. 4)LysX is suggested to modify α-amino group of α-aminoadipate in lysine biosynthetic pathway. To elucidate the mechanism for the modification, LysX from T.thermophilus HB27 and its paralogue in Sulfolobus tokodaii were crystallized. 5)Transcription of major gene cluster for lysine biosynthesis is regulated by lysine through attenuation mechanism similar to that of trp operon in Escherichia coli. In addition, the transcription is further regulated by ArgR, a transcriptional regulator in arginine biosynthesis, and PutR, a transcriptional regulator in proline metabolism.