Xue Zhixiong, McCluskey Michael, Cantera Keith, Sariaslani F Sima, Huang Lixuan
Biochemical Sciences and Engineering, DuPont CR&D, DuPont Experimental Station, Wilmington, DE 19880-0173, USA.
J Ind Microbiol Biotechnol. 2007 Sep;34(9):599-604. doi: 10.1007/s10295-007-0229-1. Epub 2007 Jun 30.
A tyrosine ammonia-lyase (TAL) enzyme from the photosynthetic bacterium Rhodobacter sphaeroides (RsTAL) was identified, cloned and functionally expressed in Escherichia coli, where conversion of tyrosine to p-hydroxycinnamic acid (pHCA) was demonstrated. The RsTAL enzyme is implicated in production of pHCA, which serves as the cofactor for synthesis of the photoactive yellow protein (PYP) in photosynthetic bacteria. The wild type RsTAL enzyme, while accepting both tyrosine and phenylalanine as substrate, prefers tyrosine, but a serendipitous RsTAL mutant identified during PCR amplification of the RsTAL gene, demonstrates much higher preference for phenylalanine as substrate and deaminates it to produces cinnamic acid. Sequence analysis showed the presence of three mutations: Met4 --> Ile, Ile325 --> Val and Val409 --> Met in this mutant. Sequence comparison with Rhodobacter capsulatus TAL (RcTAL) shows that Val409 is conserved between RcTAL and RsTAL. Two single mutants of RsTAL, Val409 --> Met and Val 409 --> Ile, generated by site-directed mutagenesis, demonstrate greater preference for phenylalanine compared to the wild type enzyme. Our studies illustrate that relatively minor changes in the primary structure of an ammonia-lyase enzyme can significantly affect its substrate specificity.
从光合细菌球形红杆菌(RsTAL)中鉴定出一种酪氨酸解氨酶(TAL),并在大肠杆菌中进行克隆和功能表达,在该菌中证实了酪氨酸可转化为对羟基肉桂酸(pHCA)。RsTAL酶与pHCA的产生有关,pHCA是光合细菌中合成光活性黄色蛋白(PYP)的辅因子。野生型RsTAL酶虽然能同时接受酪氨酸和苯丙氨酸作为底物,但更倾向于酪氨酸,不过在RsTAL基因的PCR扩增过程中偶然发现的一个RsTAL突变体,对苯丙氨酸作为底物的偏好性要高得多,并将其脱氨基生成肉桂酸。序列分析表明该突变体存在三个突变:Met4 --> Ile、Ile325 --> Val和Val409 --> Met。与荚膜红杆菌TAL(RcTAL)的序列比较表明,Val409在RcTAL和RsTAL之间是保守的。通过定点诱变产生的RsTAL的两个单突变体Val409 --> Met和Val 409 --> Ile,与野生型酶相比,对苯丙氨酸的偏好性更高。我们的研究表明,解氨酶酶一级结构中相对较小的变化会显著影响其底物特异性。