Ortega Manuel A, Hao Yue, Walker Mark C, Donadio Stefano, Sosio Margherita, Nair Satish K, van der Donk Wilfred A
Department of Biochemistry; University of Illinois at Urbana-Champaign, Roger Adams Laboratory, 600 S. Mathews Ave., Urbana IL, 61801, USA.
Department of Chemistry and Howard Hughes Medical Institute; University of Illinois at Urbana-Champaign, Roger Adams Laboratory, 600 S. Mathews Ave., Urbana IL, 61801,USA.
Cell Chem Biol. 2016 Mar 17;23(3):370-380. doi: 10.1016/j.chembiol.2015.11.017. Epub 2016 Feb 11.
Class I lantibiotic dehydratases dehydrate selected Ser/Thr residues of a precursor peptide. Recent studies demonstrated the requirement of glutamyl-tRNA(Glu) for Ser/Thr activation by one of these enzymes (NisB) from the Firmicute Lactococcus lactis. However, the generality of glutamyl-tRNA(Glu) usage and the tRNA specificity of lantibiotic dehydratases have not been established. Here we report the 2.7-Å resolution crystal structure, along with the glutamyl-tRNA(Glu) utilization of MibB, a lantibiotic dehydratase from the Actinobacterium Microbispora sp. 107891 involved in the biosynthesis of the clinical candidate NAI-107. Biochemical assays revealed nucleotides A73 and U72 within the tRNA(Glu) acceptor stem to be important for MibB glutamyl-tRNA(Glu) usage. Using this knowledge, an expression system for the production of NAI-107 analogs in Escherichia coli was developed, overcoming the inability of MibB to utilize E. coli tRNA(Glu). Our work provides evidence for a common tRNA(Glu)-dependent dehydration mechanism, paving the way for the characterization of lantibiotics from various phyla.
I类羊毛硫抗生素脱水酶可使前体肽中选定的丝氨酸/苏氨酸残基脱水。最近的研究表明,来自厚壁菌门乳酸乳球菌的其中一种此类酶(NisB)激活丝氨酸/苏氨酸需要谷氨酰tRNA(Glu)。然而,谷氨酰tRNA(Glu)使用的普遍性以及羊毛硫抗生素脱水酶的tRNA特异性尚未确定。在此,我们报告了来自放线菌微小双孢菌属107891的羊毛硫抗生素脱水酶MibB的2.7 Å分辨率晶体结构,以及其对谷氨酰tRNA(Glu)的利用情况,该酶参与临床候选药物NAI-107的生物合成。生化分析表明,tRNA(Glu)受体茎中的核苷酸A73和U72对MibB使用谷氨酰tRNA(Glu)很重要。利用这一知识,开发了一种在大肠杆菌中生产NAI-107类似物的表达系统,克服了MibB无法利用大肠杆菌谷氨酰tRNA(Glu)的问题。我们的工作为常见的依赖谷氨酰tRNA(Glu)的脱水机制提供了证据,为表征来自不同门的羊毛硫抗生素铺平了道路。