Role and dynamics of an agmatinase-like protein (AGM-1) in Neurospora crassa.,Fungal Genetics and Biology

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Role and dynamics of an agmatinase-like protein (AGM-1) in Neurospora crassa.
Fungal Genetics and Biology ( IF 2.4 ) Pub Date : 2019-08-26 , DOI: 10.1016/j.fgb.2019.103264
Luis L Pérez-Mozqueda ₁ , Rafael Vazquez-Duhalt ₂ , Ernestina Castro-Longoria ₁

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Agmatinase is known as a metalloenzyme which hydrolyzes agmatine to produce urea and putrescine, being crucial in the alternative pathway to produce polyamines. In this study, an agmatinase-like protein (AGM-1) (NCU 01348) in the filamentous fungus Neurospora crassa is reported. Purified AGM-1 from N. crassa displays enzymatic activity hydrolyzing agmatine; therefore, it can be considered as an agmatinase-like protein. However, its role in the alternative pathway to produce polyamines apparently is not its main function since only a slight reduction of polyamines concentration was detected in the Δagm-1 het strain. Moreover, the null mutant Δagm-1 (homokaryon strain) was unable to grow and the deficiency of agm-1 in the heterokaryon strain provoked a decrease in elongation rate, conidia and biomass production, despite of having de constitutive pathway via the ornithine decarboxylase (ODC). Additionally, mature hyphae of the Δagm-1 het strain presented unusual apical branching and a disorganized Spitzenkörper (Spk). Trying to reveal the role of AGM-1in N. crassa, the protein was tagged with GFP and interestingly the dynamics and intracellular localization of AGM-1 closely resembles the F-actin population. This finding was further examined in order to elucidate if AGM-1is in a close association with F-actin. Since polyamines, among them agmatine, have been reported to act as stabilizers of actin filaments, we evaluated in vitro G-actin polymerization in the presence of agmatine and the effect of purified AGM-1 from N. crassa on these polymerized actin filaments. It was found that polymerization of actin filaments increases in the presence of agmatine and the addition of purified AGM-1 from N. crassa depolymerizes these actin filaments. Also, it was determined that an intact substrate binding site of the enzyme is necessary for the localization pattern of the native AGM-1. These results suggest that in N. crassa AGM-1 has a close association with the F-actin population via its substrate agmatine, playing an essential role during cell development.

中文翻译：

芥末酶样蛋白（AGM-1）在神经孢菌中的作用和动力学。

胍丁胺酶被称为金属酶，其水解胍丁胺以产生尿素和腐胺，这在产生多胺的替代途径中是至关重要的。在这项研究中，报道了丝状真菌Neurospora crassa中的一种类固胺酶样蛋白（AGM-1）（NCU 01348）。来自N. crassa的纯化的AGM-1具有水解胍丁胺的酶活性。因此，它可以被认为是一种胍基酶样蛋白。但是，它在生产多胺的替代途径中的作用显然不是其主要功能，因为在Δagm-1het菌株中仅检测到多胺浓度略有降低。此外，无效突变体Δagm-1（纯核细胞株）无法生长，异核体株中agm-1的缺乏导致伸长率，分生孢子和生物量产生下降，尽管具有通过鸟氨酸脱羧酶（ODC）的构成性途径。此外，Δagm-1het菌株的成熟菌丝表现出不寻常的根尖分支和无序的Spitzenkörper（Spk）。为了揭示AGM-1在猪笼草中的作用，用GFP标记了该蛋白，有趣的是，AGM-1的动力学和细胞内定位与F-肌动蛋白种群非常相似。为了阐明AGM-1是否与F-肌动蛋白密切相关，进一步检查了该发现。由于据报道聚胺（其中包括胍丁胺）可作为肌动蛋白丝的稳定剂，因此我们在存在胍丁胺的情况下评估了体外G-肌动蛋白聚合反应，以及从景天猪笼草中提纯的AGM-1对这些聚合的肌动蛋白丝的影响。已经发现，在存在胍丁胺的情况下，肌动蛋白丝的聚合增加，并且从猪笼草中纯化的AGM-1的添加使这些肌动蛋白丝解聚。此外，已确定完整的酶底物结合位点对于天然AGM-1的定位模式是必需的。这些结果表明，在猪笼草中，AGM-1通过其底物胍丁胺与F-肌动蛋白群体密切相关，在细胞发育过程中起着至关重要的作用。

更新日期：2019-08-26

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