Bradley Michael E, Rest Joshua S, Li Wen-Hsiung, Schwartz Nancy B
Department of Pediatrics, University of Chicago, 5841 South Maryland Avenue, WCH C519A, Chicago, IL, 60637, USA,
J Mol Evol. 2009 Jan;68(1):1-13. doi: 10.1007/s00239-008-9181-6. Epub 2008 Dec 6.
The enzymes catalyzing the first two reactions in the sulfate activation pathway, ATP-sulfurylase (S) and APS-kinase (K), are fused as 'KS' in animals but are fused as 'SK' in select bacteria and fungi. We have discovered a novel triple fusion protein of K, S, and pyrophosphatase (P) in several protozoan genomes within the Stramenopile lineage. These triple domain fusion proteins led us to hypothesize that pyrophosphatase enzymes and sulfate activation enzymes physically interact to impact the thermodynamics of the sulfate activation pathway. In support of this hypothesis, we demonstrate through biochemical assays that separately encoded KS and P proteins physically interact and that KS/P complexes activate more sulfate than KS alone. We also conclude on the basis of phylogenetic analyses that all known KS fusions originate from a single fusion event early in the eukaryotic lineage. Strikingly, our analyses support the same conclusion for all known SK fusions. These observations indicate that the patchwork of fused and nonfused S and K genes observed in modern-day eukaryotes and prokaryotes are the result of the two ancestral fusion genes evolving by an assortment of gene fissions, duplications, deletions, and horizontal transfers in different lineages. Our integrative use of genomics, phylogenetics, and biochemistry to characterize pyrophosphatase as a new member of the sulfate activation pathway should be effective at identifying new protein members and connections in other molecular pathways.
在硫酸盐激活途径中催化前两个反应的酶，即ATP - 硫酸化酶（S）和APS - 激酶（K），在动物中融合为“KS”，但在某些细菌和真菌中融合为“SK”。我们在不等鞭毛类谱系的几个原生动物基因组中发现了一种由K、S和焦磷酸酶（P）组成的新型三联融合蛋白。这些三结构域融合蛋白使我们推测焦磷酸酶和硫酸盐激活酶在物理上相互作用，从而影响硫酸盐激活途径的热力学。为支持这一假设，我们通过生化分析证明，单独编码的KS和P蛋白在物理上相互作用，并且KS/P复合物比单独的KS激活更多的硫酸盐。我们还基于系统发育分析得出结论，所有已知的KS融合都起源于真核生物谱系早期的一次单一融合事件。引人注目的是，我们的分析对所有已知的SK融合也支持相同的结论。这些观察结果表明，在现代真核生物和原核生物中观察到的S和K基因融合与非融合的拼凑情况，是两个祖先融合基因在不同谱系中通过各种基因裂变、复制、缺失和水平转移进化的结果。我们综合运用基因组学、系统发育学和生物化学方法将焦磷酸酶鉴定为硫酸盐激活途径的一个新成员，这应该能够有效地识别其他分子途径中的新蛋白质成员和连接关系。
