Enzymological and molecular analysis of a novel multiple-substrate aminotransferase from a hyperthermophilic archaeon.

一种超嗜热古菌，深热球菌，拥有一种具有极广泛底物特异性的氨基酸转氨酶（多底物转氨酶；MsAT）。MsAT由两个均匀的45000个亚单位组成。在80℃以上、pH 6.0 ~ 8.0之间，活性最高。活性需要吡哆醛5′-磷酸。该酶催化l -丙氨酸、l -亮氨酸、l -异亮氨酸、l -缬氨酸、l -苯丙氨酸、l -酪氨酸、l -色氨酸、l -蛋氨酸、l -半胱氨酸、l -苏氨酸和l -赖氨酸的氨基转移，以2-氧戊二酸盐作为氨基受体。克隆了MsAT编码基因，并在大肠杆菌中表达。MsAT仅显示低程度的序列同源性（小于25%）到目前为止所表征的任何转氨酶。然而，在EMBL数据库中进行同源性检索发现，博氏焦球菌和solfataricus均具有与MsAT基因同源的开放阅读框。MsAT和这些同源物在基于氨基酸序列的进化树上形成了一个独立于其他转氨酶的新簇。

A hyperthermophilic archaeon, Thermococcus profundus, possessed an amino acid aminotransferase with extremely broad substrate specificity (multiple-substrate aminotransferase ; MsAT). MsAT was composed of two homogeneous subunits of 45,000. The maximal activity was detected at above 80ﾟC and between pH 6.0 and 8.0. Pyridoxal 5'-phosphate was required for the activity. The enzyme catalyzed aminotransfer from L-alanine, L-leucine, L-isoleucine, L-valine, L-phenylalanine, L-tyrosine, L-tryptophan, L-methionine, L-cystein, L-threonine and L-lysine using 2-oxoglutarate as an amino receptor.The gene encoding MsAT was cloned and expressed in Escherichia coil. MsAT revealed only a low degree of sequence identity (less than 25%) to any aminotransferases so far characterized. However, homology search against EMBL database revealed that Pyrococcus horikoshii and Sulfolobus solfataricus possessed an open reading frame homologous to the MsAT gene. MsAT and these homologues formed a novel cluster independent from other aminotransferases on an evolutionary tree based on the amino acid sequences.