2,20-Bipyridine(2,20-BiPy)is an attractive core structure present in a number of biologically active natural products,including the structurally related antibiotics caerulomycins(CAEs)and collismycins(COLs).Their bios...2,20-Bipyridine(2,20-BiPy)is an attractive core structure present in a number of biologically active natural products,including the structurally related antibiotics caerulomycins(CAEs)and collismycins(COLs).Their biosynthetic pathways share a similar key 2,20-BiPy-L-leucine intermediate,which is desulfurated or sulfurated at C5,arises from a polyketide synthase/nonribosomal peptide synthetase hybrid assembly line.Focusing on the common off-line modification steps,we here report that the removal of the“auxiliary”L-leucine residue relies on the metallo-dependent amidohydrolase activity of CaeD or ColD.This activity leads to the production of similar 2,20-BiPy carboxylate products that then receive an oxime functionality that is characteristic for both CAEs and COLs.Unlike many metallo-dependent amidohydrolase superfamily proteins that have been previously reported,these proteins(particularly CaeD)exhibited a strong zinc ion-binding capacity that was proven by site-specific mutagenesis studies to be essential to proteolytic activity.The kinetics of the conversions that respectively involve CaeD and ColD were analyzed,showing the differences in the efficiency and substrate specificity of these two proteins.These findings would generate interest in the metallo-dependent amidohydrolase superfamily proteins that are involved in the biosynthesis of bioactive natural products.展开更多
【背景】赭曲霉毒素A (ochratoxin A, OTA)是一种可以致癌的真菌毒素,其污染严重影响食品安全,危害人类健康。生物降解法去除OTA污染是近些年的研究热点,发掘高效的OTA降解脱毒酶资源具有重要的意义。【目的】筛选高效的OTA降解菌株并...【背景】赭曲霉毒素A (ochratoxin A, OTA)是一种可以致癌的真菌毒素,其污染严重影响食品安全,危害人类健康。生物降解法去除OTA污染是近些年的研究热点,发掘高效的OTA降解脱毒酶资源具有重要的意义。【目的】筛选高效的OTA降解菌株并从中克隆降解基因,为生物脱毒方法的开发提供基因和酶资源。【方法】利用OTA为唯一碳源的筛选培养基从土壤中筛选纯化OTA降解菌株,通过16SrRNA基因序列分析确定其分类地位,利用高效液相色谱(high performanceliquidchromatography,HPLC)分析其降解产物。通过同源序列比对的方法克隆降解基因并与载体pET-29a(+)相连,然后在大肠杆菌BL21(DE3)中表达。利用Ni2+亲和层析对表达产物进行纯化,研究其对OTA的降解活性和酶学特征。【结果】筛选到一株高效的OTA降解菌株,在12h内能够完全降解1μg/mL的OTA;初步鉴定该菌株属于Niastella,编号为JX-6;菌株JX-6通过酰胺键断裂途径降解OTA生成无毒的OTα;从菌株JX-6中鉴定了一个OTA酰胺水解酶,命名为NcOTase;NcOTase与已报道的OTA酰胺水解酶序列相似性较低,仅为31%–53%;纯化的NcOTase具有OTA水解活性,比酶活为60.3U/mg,活性显著高于大部分已报道的OTA降解酶。【结论】NcOTase是一个高效的OTA降解脱毒酶,在去除食品和饲料中OTA污染方面具有很好的应用前景。展开更多
基金This workwas supported in part by grants from NSFC(21472231,21520102004,31430005,and 81302674)CAS(SQYZDJ-SSWSLH1037 and XDB20020200)+1 种基金STCM(14JC1407700 and 15JC1400400)K.C.Wong Education Foundation and Chang-Jiang Scholars Program of China.
文摘2,20-Bipyridine(2,20-BiPy)is an attractive core structure present in a number of biologically active natural products,including the structurally related antibiotics caerulomycins(CAEs)and collismycins(COLs).Their biosynthetic pathways share a similar key 2,20-BiPy-L-leucine intermediate,which is desulfurated or sulfurated at C5,arises from a polyketide synthase/nonribosomal peptide synthetase hybrid assembly line.Focusing on the common off-line modification steps,we here report that the removal of the“auxiliary”L-leucine residue relies on the metallo-dependent amidohydrolase activity of CaeD or ColD.This activity leads to the production of similar 2,20-BiPy carboxylate products that then receive an oxime functionality that is characteristic for both CAEs and COLs.Unlike many metallo-dependent amidohydrolase superfamily proteins that have been previously reported,these proteins(particularly CaeD)exhibited a strong zinc ion-binding capacity that was proven by site-specific mutagenesis studies to be essential to proteolytic activity.The kinetics of the conversions that respectively involve CaeD and ColD were analyzed,showing the differences in the efficiency and substrate specificity of these two proteins.These findings would generate interest in the metallo-dependent amidohydrolase superfamily proteins that are involved in the biosynthesis of bioactive natural products.
文摘【背景】赭曲霉毒素A (ochratoxin A, OTA)是一种可以致癌的真菌毒素,其污染严重影响食品安全,危害人类健康。生物降解法去除OTA污染是近些年的研究热点,发掘高效的OTA降解脱毒酶资源具有重要的意义。【目的】筛选高效的OTA降解菌株并从中克隆降解基因,为生物脱毒方法的开发提供基因和酶资源。【方法】利用OTA为唯一碳源的筛选培养基从土壤中筛选纯化OTA降解菌株,通过16SrRNA基因序列分析确定其分类地位,利用高效液相色谱(high performanceliquidchromatography,HPLC)分析其降解产物。通过同源序列比对的方法克隆降解基因并与载体pET-29a(+)相连,然后在大肠杆菌BL21(DE3)中表达。利用Ni2+亲和层析对表达产物进行纯化,研究其对OTA的降解活性和酶学特征。【结果】筛选到一株高效的OTA降解菌株,在12h内能够完全降解1μg/mL的OTA;初步鉴定该菌株属于Niastella,编号为JX-6;菌株JX-6通过酰胺键断裂途径降解OTA生成无毒的OTα;从菌株JX-6中鉴定了一个OTA酰胺水解酶,命名为NcOTase;NcOTase与已报道的OTA酰胺水解酶序列相似性较低,仅为31%–53%;纯化的NcOTase具有OTA水解活性,比酶活为60.3U/mg,活性显著高于大部分已报道的OTA降解酶。【结论】NcOTase是一个高效的OTA降解脱毒酶,在去除食品和饲料中OTA污染方面具有很好的应用前景。
