Lehmann Christopher, Doseeva Victoria, Pullalarevu Sadhana, Krajewski Wojciech, Howard Andrew, Herzberg Osnat
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, Maryland 20850, USA.
Proteins. 2004 Aug 1;56(2):376-83. doi: 10.1002/prot.20103.
The Escherichia coli open reading frame YbdK encodes a member of a large bacterial protein family of unknown biological function. The sequences within this family are remotely related to the sequence of gamma-glutamate-cysteine ligase (gamma-GCS), an enzyme in the glutathione biosynthetic pathway. A gene encoding gamma-GCS in E. coli is already known. The 2.15 A resolution crystal structure of YbdK reveals an overall fold similar to that of glutamine synthetase (GS), a nitrogen metabolism enzyme that ligates glutamate and ammonia to yield glutamine. GS and gamma-GCS perform related chemical reactions and require ATP and Mg2+ for their activity. The Mg2+-dependent binding of ATP to YbdK was confirmed by fluorescence spectroscopy employing 2'(or 3')-O-(trinitrophenyl)adenosine 5'-triphosphate, and yielding a dissociation constant of 3 +/- 0.5 microM. The structure of YbdK contains a crevice that corresponds to the binding sites of ATP, Mg2+ and glutamate in GS. Many of the GS residues that coordinate the metal ions and interact with glutamic acid and the phosphoryl and ribosyl groups of ATP are also present in YbdK. GS amino acids that have been associated with ammonia binding have no obvious counterparts in YbdK, consistent with a substrate specificity that is different from that of GS. Ligase activity between glutamic acid and each of the twenty amino acid residues was tested on high performance liquid chromatography (HPLC) by following the hydrolysis of ATP to ADP. Catalysis was observed only with cysteine. A pyruvate kinase/lactic acid dehydrogenase coupled assay was used to rule out GS activity and to determine that YbdK exhibits gamma-GCS activity. The catalytic rate was found to be approximately 500-fold slower than that reported for authentic gamma-GCS.
大肠杆菌开放阅读框YbdK编码一个大型细菌蛋白家族的成员，该家族的生物学功能未知。这个家族中的序列与γ-谷氨酰胺半胱氨酸连接酶（γ-GCS）的序列有远缘关系，γ-GCS是谷胱甘肽生物合成途径中的一种酶。大肠杆菌中编码γ-GCS的基因已为人所知。YbdK的2.15埃分辨率晶体结构显示其整体折叠与谷氨酰胺合成酶（GS）相似，GS是一种氮代谢酶，可将谷氨酸和氨连接生成谷氨酰胺。GS和γ-GCS进行相关的化学反应，并且其活性需要ATP和Mg2+。通过使用2'（或3'）-O-（三硝基苯基）腺苷5'-三磷酸的荧光光谱法证实了ATP与YbdK的Mg2+依赖性结合，解离常数为3±0.5微摩尔。YbdK的结构包含一个裂隙，对应于GS中ATP、Mg2+和谷氨酸的结合位点。许多在GS中与金属离子配位并与谷氨酸以及ATP的磷酸基和核糖基相互作用的残基在YbdK中也存在。与氨结合相关的GS氨基酸在YbdK中没有明显的对应物，这与不同于GS的底物特异性一致。通过跟踪ATP水解为ADP，在高效液相色谱（HPLC）上测试了谷氨酸与二十种氨基酸残基中每一种之间的连接酶活性。仅在半胱氨酸存在下观察到催化作用。使用丙酮酸激酶/乳酸脱氢酶偶联测定法排除GS活性，并确定YbdK具有γ-GCS活性。发现催化速率比真实的γ-GCS报道的速率慢约500倍。
