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首页> 外文期刊>Biochimica et biophysica acta: BBA: International journal of biochemistry, biophysics and molecular biololgy. Proteins and Proteomics >The first archaeal agmatinase from anaerobic hyperthermophilic archaeon Pyrococcus horikoshii: cloning, expression, and characterization
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The first archaeal agmatinase from anaerobic hyperthermophilic archaeon Pyrococcus horikoshii: cloning, expression, and characterization

机译：厌氧性超嗜热古生热球菌的第一个古细菌胍丁胺酶：克隆，表达和鉴定

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摘要

Agmatinase is one of the key enzymes in the biosynthesis of polyamines such as putrescine and sperimidine from arginine in microorganisms. The gene (PH0083) encoding the putative agmatinase of hyperthermophilic archaeon Pyrococcus horikoshii was identified based on the genome database. The gene was cloned and expressed, and the product was mainly obtained as inactive inclusion body in Escherichia coli. The inclusion body was dissolved in 6 M guanidine–HCl and successively refolded to active enzyme by the dilution of the denaturant. The enzyme exclusively catalyzed the hydrolysis of agmatine, but not arginine. This indicates that PH0083 codes agmatinase. The enzyme required divalent cations such as Co2+, Ca2+ and Mn2+ for the activity. The highest activity was observed under fairly alkaline conditions, like pH 11. The purified recombinant enzyme consisted of four identical subunits with a molecular mass of 110–145 kDa. The enzyme was extremely thermostable: the full activity was retained on heating at 80 °C for 10 min, and a half of the activity was retained by incubation at 90 °C for 10 min. From a typical Michaelis–Menten type kinetics, an apparent Km value for agmatine was determined to be 0.53 mM. Phylogenic analysis revealed that the agmatinase from P. horikoshii does not belong to any clusters of enzymes found in bacteria and eukarya. This is the first description of the presence of archaeal agmatinase and its characteristics.
机译：精氨酸酶是在微生物中由精氨酸生物合成多胺如腐胺和亚精胺的关键酶之一。基于基因组数据库鉴定了编码超嗜热古生毕赤酵母的假定胍丁胺酶的基因（PH0083）。克隆并表达了该基因，该产物主要以大肠杆菌中的非活性包涵体形式获得。包涵体溶解在6 M盐酸胍中，并通过稀释变性剂依次折叠成活性酶。该酶仅催化胍丁胺的水解，但不催化精氨酸的水解。这表明PH0083编码胍丁胺酶。该酶需要二价阳离子如Co2 +，Ca2 +和Mn2 +才能发挥活性。在相当碱性的条件下，例如pH 11，观察到了最高的活性。纯化的重组酶由四个相同的亚基组成，分子量为110–145 kDa。酶极热稳定：在80°C加热10分钟后，其全部活性得以保留，而在90°C孵育10分钟，则可保留一半的活性。根据典型的Michaelis-Menten型动力学，胍丁胺的表观Km值确定为0.53 mM。系统发育分析表明，来自日本假单胞菌的胍丁胺酶不属于细菌和真核生物中发现的任何酶簇。这是古细菌性胍丁胺糖酶及其特性的首次描述。

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