像真菌和一些原核生物,植物使用噻唑合酶(THI4)来制造硫胺素的噻唑前体。真菌THI4s是自杀酶,可破坏必需的活性位点Cys残基以获得噻唑形成所需的硫原子。相比之下,某些原核THI4s没有活性位点Cys,使用硫化物作为硫供体,是真正的催化剂。植物THI4s中保守活性位点Cys的存在和其他间接证据表明它们是自杀的。为了证实这一点,我们补充了拟南芥-1突变体,缺乏THI4活性,带有His标记的拟南芥THI4构建体。从叶子中提取的THI4胰蛋白酶肽的LC-MS分析表明,活性位点Cys主要是脱硫形式,与植物中具有自杀机制的THI4一致。出乎意料的是,转录组数据挖掘和深度蛋白质组分析表明,大麦,小麦,和燕麦都具有广泛表达的经典THI4,具有活性位点Cys,和类似THI4的类似物(非CysTHI4),没有活性位点Cys,是发育中谷物中THI4的主要类型。转录组学证据还表明大麦,小麦,燕麦谷物从头合成硫胺素,暗示他们的非CysTHI4s合成噻唑。结构建模支持这种推断,正如所证明的那样,非CysTHI4具有显着的能力来补充大肠杆菌中的噻唑营养缺陷型。因此,有一个初步证据表明,非Cys谷物THI4s,像它们的原核生物一样,是催化噻唑合酶。生物能量计算表明,相对于自杀THI4s,这种酶可以在灌浆期节省大量的能量。
Like fungi and some prokaryotes, plants use a thiazole synthase (THI4) to make the thiazole precursor of
thiamin. Fungal THI4s are suicide enzymes that destroy an essential active-site Cys residue to obtain the sulfur atom needed for thiazole formation. In contrast, certain prokaryotic THI4s have no active-site Cys, use sulfide as sulfur donor, and are truly catalytic. The presence of a conserved active-site Cys in plant THI4s and other indirect evidence implies that they are suicidal. To confirm this, we complemented the Arabidopsistz-1 mutant, which lacks THI4 activity, with a His-tagged Arabidopsis THI4 construct. LC-MS analysis of tryptic peptides of the THI4 extracted from leaves showed that the active-site Cys was predominantly in desulfurated form, consistent with THI4 having a suicide mechanism in planta. Unexpectedly, transcriptome data mining and deep proteome profiling showed that barley, wheat, and oat have both a widely expressed canonical THI4 with an active-site Cys, and a THI4-like paralog (non-Cys THI4) that has no active-site Cys and is the major type of THI4 in developing grains. Transcriptomic evidence also indicated that barley, wheat, and oat grains synthesize
thiamin de novo, implying that their non-Cys THI4s synthesize thiazole. Structure modeling supported this inference, as did demonstration that non-Cys THI4s have significant capacity to complement thiazole auxotrophy in Escherichia coli. There is thus a prima facie case that non-Cys cereal THI4s, like their prokaryotic counterparts, are catalytic thiazole synthases. Bioenergetic calculations show that, relative to suicide THI4s, such enzymes could save substantial energy during the grain-filling period.