Williams J, Phillips A L, Gaskin P, Hedden P
IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS41 9AF, United Kingdom.
Plant Physiol. 1998 Jun;117(2):559-63. doi: 10.1104/pp.117.2.559.
cDNA corresponding to the GA4 gene of Arabidopsis thaliana L. (Heynh. ) was expressed in Escherichia coli, from which cell lysates converted [14C]gibberellin (GA)9 and [14C]GA20 to radiolabeled GA4 and GA1, respectively, thereby confirming that GA4 encodes a GA 3beta-hydroxylase. GA9 was the preferred substrate, with a Michaelis value of 1 microm compared with 15 microm for GA20. Hydroxylation of these GAs was regiospecific, with no indication of 2beta-hydroxylation or 2,3-desaturation. The capacity of the recombinant enzyme to hydroxylate a range of other GA substrates was investigated. In general, the preferred substrates contained a polar bridge between C-4 and C-10, and 13-deoxy GAs were preferred to their 13-hydroxylated analogs. Therefore, no activity was detected using GA12-aldehyde, GA12, GA19, GA25, GA53, or GA44 as the open lactone (20-hydroxy-GA53), whereas GA15, GA24, and GA44 were hydroxylated to GA37, GA36, and GA38, respectively. The open lactone of GA15 (20-hydroxy-GA12) was hydroxylated but less efficiently than GA15. In contrast to the free acid, GA25 19,20-anhydride was 3beta-hydroxylated to give GA13. 2,3-Didehydro-GA9 and GA5 were converted by recombinant GA4 to the corresponding epoxides 2, 3-oxido-GA9 and GA6.
拟南芥L.（海因）GA4基因对应的cDNA在大肠杆菌中表达，其细胞裂解物分别将[14C]赤霉素（GA）9和[14C]GA20转化为放射性标记的GA4和GA1，从而证实GA4编码一种GA 3β-羟化酶。GA9是首选底物，米氏值为1微摩尔，而GA20为15微摩尔。这些GA的羟基化具有区域特异性，没有2β-羟基化或2,3-去饱和的迹象。研究了重组酶对一系列其他GA底物进行羟基化的能力。一般来说，首选底物在C-4和C-10之间含有一个极性桥，并且13-脱氧GA比其13-羟基化类似物更受青睐。因此，使用GA12-醛、GA12、GA19、GA25、GA53或GA44作为开环内酯（20-羟基-GA53）时未检测到活性，而GA15、GA24和GA44分别被羟基化为GA37、GA36和GA38。GA15的开环内酯（20-羟基-GA12）被羟基化，但效率低于GA15。与游离酸相反，GA25 19,20-酸酐被3β-羟基化生成GA13。2,3-二脱氢-GA9和GA5被重组GA4转化为相应的环氧化物2,3-环氧-GA9和GA6。
