Nakamura Hiro, Kumita Hideyuki, Imai Kiyohiro, Iizuka Tetsutaro, Shiro Yoshitsugu
RIKEN Harima Institute/SPring-8, Mikazuki, Sayo, Hyogo 679-5148, Japan.
Proc Natl Acad Sci U S A. 2004 Mar 2;101(9):2742-6. doi: 10.1073/pnas.0305795101. Epub 2004 Feb 17.
The rhizobial FixL/FixJ system, a paradigm of heme-based oxygen sensors, belongs to the ubiquitous two-component signal transduction system. Oxygen-free (deoxy) FixL is autophosphorylated at an invariant histidine residue by using ATP and catalyzes the concomitant phosphoryl transfer to FixJ, but oxygen binding to the FixL heme moiety inactivates the kinase activity. Here we demonstrate that ADP acts as an allosteric effector, reducing the oxygen-binding affinity of the sensor domain in FixL when it is produced from ATP in the kinase reaction. The addition of ADP to a solution of purified wild-type FixL resulted in an approximately 4- to 5-fold decrease in oxygen-binding affinity in the presence of FixJ. In contrast, phosphorylation-deficient mutants, in which the well conserved ATP-binding catalytic site of the kinase domain is impaired, showed no such allosteric effect. This discovery casts light on the significance of homodimerization of two-component histidine kinases; ADP, generated in the phosphorylation reaction in one subunit of the homodimer, enhances the histidine kinase activity of the other, analogous to a two-cylinder reciprocating engine by reducing the ligand-binding affinity.
根瘤菌FixL/FixJ系统是基于血红素的氧传感器的典范，属于普遍存在的双组分信号转导系统。无氧（脱氧）的FixL利用ATP在一个不变的组氨酸残基上进行自身磷酸化，并催化伴随的磷酸基转移至FixJ，但氧与FixL血红素部分的结合会使激酶活性失活。在此我们证明，ADP作为变构效应物，在激酶反应中由ATP生成时，会降低FixL中传感器结构域的氧结合亲和力。在存在FixJ的情况下，向纯化的野生型FixL溶液中添加ADP会导致氧结合亲和力降低约4至5倍。相比之下，激酶结构域中保守的ATP结合催化位点受损的磷酸化缺陷突变体则未表现出这种变构效应。这一发现揭示了双组分组氨酸激酶同二聚化的重要性；同二聚体一个亚基的磷酸化反应中产生的ADP会增强另一个亚基的组氨酸激酶活性，类似于双缸往复式发动机通过降低配体结合亲和力来实现。
