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Urease Inhibition in the Presence of N-(n-Butyl)thiophosphoric Triamide, a Suicide Substrate: Structure and Kinetics
Biochemistry ( IF 2.9 ) Pub Date : 2017-09-15 00:00:00 , DOI: 10.1021/acs.biochem.7b00750
Luca Mazzei 1 , Michele Cianci 2 , Umberto Contaldo 1 , Francesco Musiani 1 , Stefano Ciurli 1
Affiliation  

The nickel-dependent enzyme urease is a virulence factor for a large number of pathogenic and antibiotic-resistant bacteria, as well as a negative factor for the efficiency of soil nitrogen fertilization for crop production. The use of urease inhibitors to offset these effects requires knowledge, at a molecular level, of their mode of action. The 1.28 Å resolution structure of the enzyme–inhibitor complex obtained upon incubation of Sporosarcina pasteurii urease with N-(n-butyl)thiophosphoric triamide (NBPT), a molecule largely utilized in agriculture, reveals the presence of the monoamidothiophosphoric acid (MATP) moiety, obtained upon enzymatic hydrolysis of the diamide derivative of NBPT (NBPD) to yield n-butyl amine. MATP is bound to the two Ni(II) ions in the active site of urease using a μ2-bridging O atom and terminally bound O and NH2 groups, with the S atom of the thiophosphoric amide pointing away from the metal center. The mobile flap modulating the size of the active site cavity is found in the closed conformation. Docking calculations suggest that the interaction between urease in the open flap conformation and NBPD involves a role for the conserved αArg339 in capturing and orienting the inhibitor prior to flap closure. Calorimetric and spectrophotometric determinations of the kinetic parameters of this inhibition indicate the occurrence of a reversible slow inhibition mode of action, characterized, for both bacterial and plant ureases, by a very small value of the dissociation constant of the urease–MATP complex. No need to convert NBPT to its oxo derivative NBPTO, as previously proposed, is necessary for urease inhibition.

中文翻译:

N-n-丁基)硫代磷酸三酰胺,一种自杀基质的脲酶抑制作用:结构和动力学

镍依赖性酶脲酶是大量致病性和抗生素抗性细菌的致病因子,也是土壤氮肥用于农作物生产的效率的不利因素。使用脲酶抑制剂来抵消这些作用需要在分子水平上了解其作用方式。将巴斯德曲霉脲酶与N-n-丁基)硫代磷酸三酰胺(NBPT)孵育后获得的酶抑制剂复合物的1.28Å拆分结构揭示了单酰胺基硫代磷酸(MATP)部分的存在经酶水解NBPT的二酰胺衍生物(NBPD)得到n丁胺。MATP使用μ绑定到所述两个镍(II)离子在脲酶的活性位点2 -bridging的O原子和末端结合的O和NH 2基团,硫代磷酸酰胺的S原子指向远离金属中心的位置。在闭合构象中发现了可调节活动部位腔大小的活动瓣。对接计算表明,开放性皮瓣构象中的脲酶与NBPD之间的相互作用涉及保守的αArg339在皮瓣关闭前捕获和定向抑制剂方面的作用。量热法和分光光度法测定这种抑制的动力学参数,表明存在可逆的缓慢抑制作用模式,对于细菌和植物脲酶而言,其特征是脲酶-MATP复合物的解离常数很小。如前所述,无需将NBPT转化为其含氧衍生物NBPTO即可抑制尿素酶。
更新日期:2017-09-15
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