A Novel CO-producing Metalloenzyme

描述(由申请人提供)：蛋氨酸是活细胞中烷基化等效物的重要来源。作为S-腺苷甲硫氨酸，蛋氨酸的末端甲基被用于体内广泛的转甲基化反应，乙基甘氨酸部分在快速增殖的细胞中供给多胺的生物合成。许多人类癌细胞系是蛋氨酸依赖的，而SAM代谢的产物甲硫腺苷(MTA)是多胺生物合成的有效反馈抑制物，进而阻止DNA复制和细胞周期的延续，这引起了人们对蛋氨酸代谢命运的兴趣。有证据表明，在蛋氨酸依赖的癌细胞系中，负责将S-OH3基团从MTA循环到蛋氨酸的蛋氨酸挽救途径存在缺陷。 本研究对肺炎克雷伯菌甲硫氨酸回收途径中一种重要的金属酶--酸性还原内酯双加氧酶(ARD)的结构和功能进行了深入研究。根据与酶活性部位结合的金属离子的不同，ARD可以与同一底物催化两种不同的反应。含有单一Ni+2离子的ARD在旁路分流中催化蛋氨酸回收途径中的高级中间体酸性还原内酯氧化裂解成β-甲硫基丙酸酯、甲酸盐和一氧化碳(CO)。如果结合的是Fe+2离子而不是镍(ARD‘)，则催化生成甲酸盐和蛋氨酸的酮酸前体α-酮-伽马硫代丁酸酯的反应。最近用核磁共振方法解决了镍结合ARD的结构，机理研究为ARD催化反应的自由基机理提供了证据。 拟议工作的总体目标包括：(1)确定ARD‘的溶液结构，并分析导致ARD和ARD’不同活性的结构差异。将进行定点突变、酶活性和动力学测量以及稳定的酶-底物复合体的核磁共振研究。(2)水稻和人类ARD同源基因的表达、分离、纯化及鉴定。特别是，将寻找体外和体内ARD活性的证据(即CO产生)，因为现在有相当多的证据表明CO可以作为信号分子和细胞凋亡(程序性细胞死亡)的抑制因子。

DESCRIPTION (provided by applicant): Methionine is an important source of alkylating equivalents in living cells. As S-adenosylmethionine (SAM), the terminal methyl group of methionine is used in a wide array of transmethylation reactions in vivo, and the ethylglycine moiety feeds into polyamine biosynthesis in rapidly proliferating cells. Interest in the metabolic fate of methionine is raised by the observation that many human cancer cell lines are methionine dependent, and that methylthioadenosine (MTA), a product of SAM metabolism, is a potent feedback inhibitor of polyamine biosynthesis, which in turn stops DNA replication and prevents continuation of the cell cycle. There is evidence suggesting that the methionine salvage pathway, which is responsible for recycling the S-OH3 group from MTA to methionine, is defective in methionine-dependent cancer cell lines. This proposal describes an in-depth investigation into the structure and function of a remarkable metalloenzyme from the methionine salvage pathway of the bacterium Klebsiella pneumoniae, acireductone dioxygenase (ARD). ARD can catalyze two different reactions with the same substrate depending upon the metal ion that is bound to the enzyme active site. ARD containing a single Ni+2 ion catalyzes the oxidative cleavage of acireductone, an advanced intermediate in the methionine salvage pathway, into beta-methylthiopropionate, formate and carbon monoxide (CO) in an off-pathway shunt. If a Fe+2 ion is bound instead of nickel (ARD'), the on-pathway reaction leading to formate and the ketoacid precursor of methionine, alpha-keto-gamma-methylthiobutyrate is catalyzed. A structure for Ni-bound ARD has recently been solved by NMR methods, and mechanistic studies have provided evidence for a radical mechanism for the reaction catalyzed by ARD. The general aims of the proposed work include: (1) Determination of the solution structure of ARD' and an analysis of the structural differences between ARD and ARD' that lead to their different activities. Site-directed mutagenesis, enzyme activity and kinetics measurements and NMR studies of stable enzyme-substrate complexes will be performed. (2) Expression, isolation, purification and characterization of ARD homologues from two eukaryotes, rice (Oriza sativa) and humans (Homo sapiens). In particular, evidence for in-vitro and in-vivo ARD activity will be sought (i.e., CO production), since there is now considerable evidence that CO can act as a signaling molecule and an inhibitor of apoptosis (programmed cell death).