维生素K2合成中UbiA与MenA协同调控机制研究

As a kind of essential vitamins for human body, vitamin K2 has many important physiological functions such as promote the generation of prothrombin, increase synthesis of osteocalcin, repair the mitochondria of damaged cells. Thus it has a very broad application prospects in food industry. Decrease the activity of 4-hydroxybenzoic acid oligoprenyltransferase (UbiA) combining with increase the activity of 1,4-dihydroxy-2-naphthoicacid oligoprenyltransferase (MenA) in Bacillus subtils natto can significantly promote the synthesis of vitamin K2. In this project, the fusion expression stain of UbiA and MenA with the fluorescent protein was constructed, the interaction between UbiA and MenA in vivo was revealed by fluorescence resonance energy transfer (FRET). The UbiA and MenA was purified, and the interaction between UbiA and MenA in vitro would be clarified by the surface plasmon resonance technology (SPR). The key binding sites in active area of UbiA and MenA were also being analyzed by directed evolution and site directed mutagenesis. The mechanism of synergistic reaction between UbiA and MenA was discussed respectively in cell, protein and nucleotide levels. It provides sufficient theoretical basis for regulation the synthesis of vitamin K2 in Bacillus subtils natto.

作为一种能够促进凝血酶原产生、增加骨骼密度以及修复损伤细胞线粒体的人体必需维生素，维生素K2在食品行业具有非常广阔的应用前景。纳豆芽孢杆菌中4-羟苯甲酸-聚异戊二烯转移酶（UbiA）活性的下调协同1,4-二羟基-2-萘甲酸-聚异戊二烯转移酶（MenA）活性的上调能够显著促进维生素K2的合成。为揭示UbiA与MenA的协同调控机制，本研究拟分别构建UbiA和MenA与荧光蛋白融合表达菌株，利用荧光共振能量转移技术（FRET）揭示UbiA与MenA胞内相互作用，并通过表面等离子共振技术（SPR）解析二者的胞外互作特性，进而阐明UbiA与MenA相互作用机制，同时结合定向进化和定点突变分析UbiA和MenA活性区域上的关键结合位点。分别从细胞、蛋白和核苷酸三个水平探讨UbiA与MenA的协同调控促进维生素K2合成的机制，为纳豆芽孢杆菌维生素K2合成调控提供充分的理论依据。

作为一种能够促进凝血酶原产生、增加骨骼密度以及修复损伤细胞线粒体的人体必需维生素，维生素K2在食品行业具有非常广阔的应用前景。4-羟苯甲酸-聚异戊二烯转移酶（UbiA）活性的下调协同1,4-二羟基-2-萘甲酸-聚异戊二烯转移酶（MenA）活性的上调能够显著促进维生素K2的合成。为揭示UbiA与MenA的协同调控机制，本研究分别构建UbiA和MenA与荧光蛋白融合表达菌株，利用荧光共振能量转移技术（FRET）揭示UbiA与MenA胞内相互作用，同时结合定向进化和定点突变分析UbiA和MenA活性区域上的关键结合位点。结果发现：MenA属于亲脂且疏水的膜结合型蛋白质，MenA的34-278位的氨基酸组成典型的UbiA结构功能域，且74-84和204-212氨基酸中分别存在2个NxxxDxxxxxD和NNxxDxxxD的Asp-rich motifs结构；UbiA由9个跨膜螺旋和一个膜外帽子构成活性中心腔，该中心腔是带有活性位点的活性区域。活性区域中有一个侧链GSPP结合位点和一个PHB结合位点。为便于膜内催化，UbiA有一个不寻常的活性位点侧向延伸至磷脂双分子层。如果将4-羟苯甲酸-聚异戊二烯转移酶（UbiA）的第198位天冬氨酸定点突变为丙氨酸，共表达dxr和menA，以及添加丙酮酸钠(SPY)和莽草酸(SA)，能够将MK的产量提高397%。并且，当共表达dxr, menA和ubiE，以及添加丙酮酸钠(SPY)和莽草酸(SA)后，MK的产量又有了145%的增长。因此，总体上来说，当通过定点突变削弱了UbiA酶学活性，与此同时过量表达dxr, menA和ubiE，以及添加丙酮酸钠(SPY)和莽草酸(SA)后，维生素K2代谢合成的能力提高了11倍多。这对于促进维生素K2的工业化应用有着重要的意义。