We have identified and cloned five auxin conjugate amidohydrolases (M20D peptidases) in four different hornwort species (Phaeoceros carolinianus, Megaceros tosanus, Megaceros vincentianus, and Paraphymatoceros hallii). Sequence analysis suggests that all five enzymes have greater than 60% overall similarity to tracheophyte amidohydrolases. Phylogenetic analysis supports the hypothesis that the bryophyte and tracheophyte hydrolases are derived from a common ancestor. Enzyme studies of hornwort auxin amidohydrolases all demonstrate greater activity and substrate recognition than the more ancient liverwort hydrolase (MpILR1). The mean wild-type hornwort hydrolytic activity (23.2 ± 6.8 pmol auxin released/min/ml), although higher than the liverwort activity (1.3 ± 1.1 pmol auxin released/min/ml), was almost a magnitude lower than the average activity in tracheophyte hydrolases (186.2 ± 52.1 pmol auxin released/min/ml). Two hornwort orthologues from M. vincentianus and M. tosanus possess a Glycine238/240 replacing the tracheophytically conserved Serine209, while two from P. hallii and P. carolinianus have an Alanine238 at that homologous residue location. Further enzymatic studies and three-dimensional structural analyses of the hornwort enzymes present supporting evidence that the Ala238-line of hornworts is the likely clade from which tracheophytes arose.

我们已经在四种不同的金角藻属（Phaeoceros carolinianus、Megaceros tosanus、Megaceros vincentianus和Paraphymatoceroshallii）中鉴定并克隆了五种生长素共轭酰胺水解酶（M20D 肽酶））。序列分析表明，所有五种酶与气管植物酰胺水解酶的总体相似性大于 60%。系统发育分析支持苔藓植物和气管植物水解酶源自共同祖先的假设。金鱼草生长素酰胺水解酶的酶研究都证明了比更古老的地衣水解酶 (MpILR1) 更高的活性和底物识别能力。野生型金鱼草的平均水解活性（23.2 ± 6.8 pmol 生长素释放/min/ml），虽然高于地草活性（1.3 ± 1.1 pmol 生长素释放/min/ml），但几乎比平均活性低一个数量级气管植物水解酶（186.2 ± 52.1 pmol 生长素释放/分钟/毫升）。来自M. vincentianus和M. tosanus 的两个金鱼草直系同源物具有一个Glycine238 / 240替换tracheophytically保守Serine209，而两位来自P.霍氏真杆菌和P. carolinianus具有Alanine238在该同源残基位置。金鱼草酶的进一步酶学研究和三维结构分析提供了支持证据，即金鱼草的 Ala238 系可能是气管植物产生的进化枝。