Characterization of a bifunctional PutA homologue from Bradyrhizobium japonicum and identification of an active site residue that modulates proline reduction of the flavin adenine dinucleotide cofactor
Characterization of a bifunctional PutA homologue from Bradyrhizobium japonicum and identification of an active site residue that modulates proline reduction of the flavin adenine dinucleotide cofactor
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DOI：
10.1021/bi050629k
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发表时间：
2005-06-28
期刊：
影响因子：
2.9
通讯作者：
Becker, DF
中科院分区：
文献类型：
--
作者：
Krishnan, N;Becker, DF
PutA是细菌中的双功能黄素酶，其催化脯氨酸的四电子氧化为谷氨酸。在某些原核生物如大肠杆菌中，PutA也是脯氨酸利用（put）基因的转录抑制因子，因此是三功能的。在这项工作中，我们已经开始评估双功能和三功能PutA酶之间的差异，通过检查PutA蛋白从慢生型大豆根瘤菌（BjPutA）。一级结构分析表明BjPutA缺乏E. coli PutA（EcPutA）。与此预测一致，纯化的BjPutA在天然凝胶迁移率变动测定中不表现出DNA结合活性，其中putA基因的启动子区域来自B。山茱萸表征了BjPutA的催化和氧化还原性质，并测定了BjPutA中结合的FAD/FADH（2）对的还原电位（E-m）值为-0.132 V（pH 7.5），其比EcPutA结合的FAD的E-m显著更负（类似于55 mV）。更负的E值会限制BjPutA中FAD辅因子的脯氨酸还原。在磷脂的存在下，刺激BjPutA的还原，表明脂质影响FAD氧化还原环境。因此，在极性脂质的存在下，确定BjPutA结合的FAD的E-m值为-0.114V（pH 7.5）。通过定点突变探索了BjPutA中FAD相对于EcPutA的较低还原电位的分子基础。BjPutA和EcPutA之间的氨基酸序列比对表明FAD的异咯嗪环附近的活性位点残基仅有一个差异：EcPutA中的Va 1402在BjPutA中的类似位置处被Ala 310取代。在BjPutA突变体A310 V中用瓦尔替换A310使结合的FAD相对于野生型BjPutA的还原电位升高至-0.09 V（pH 7.5）的E-m值。BjPutA突变体A310 V的电位的> 40-mV的正向偏移表明EcPutA中相应的瓦尔残基有助于平衡FAD氧化还原电位，以进行生物学上有利的脯氨酸还原，从而允许EcPutA通过脯氨酸可用性有效地调节。BjPutA在还原条件下的有限蛋白水解显示FAD还原不影响BjPutA构象，进一步表明用EcPutA观察到的氧化还原依赖性调节可能限于三功能PutA同系物。
PutA is a bifunctional flavoenzyme in bacteria that catalyzes the four-electron oxidation of proline to glutamate. In certain prokaryotes such as Escherichia coli, PutA is also a transcriptional repressor of the proline utilization (put) genes and thus is trifunctional. In this work, we have begun to assess differences between bifunctional and trifunctional PutA enzymes by examining the PutA protein from Bradyrhizobium japonicum (BjPutA). Primary structure analysis of BjPutA shows it lacks the DNA-binding domain of E. coli PutA (EcPutA). Consistent with this prediction, purified BjPutA does not exhibit DNA-binding activity in native gel mobility shift assays with promoter regions of the putA gene from B. japonicum. The catalytic and redox properties of BjPutA were characterized and a reduction potential (E-m) value of -0.132 V (pH 7.5) was determined for the bound FAD/FADH(2) couple in BjPutA that is significantly more negative (similar to 55 mV) than the E-m for EcPutA-bound FAD. The more negative E value thermodynamically limits proline reduction of the FAD cofactor in BjPutA. In the presence of phospholipids, reduction of BjPutA is stimulated, suggesting lipids influence the FAD redox environment. Accordingly, an E-m value of -0.114 V (pH 7.5) was determined for BjPutA-bound FAD in the presence of polar lipids. The molecular basis for the lower reduction potential of FAD in BjPutA relative to EcPutA was explored by site-directed mutagenesis. Amino acid sequence alignment between BjPutA and EcPutA indicates only one difference in active site residues near the isoalloxazine ring of FAD: Va1402 in EcPutA is substituted at the analogous position in BjPutA with Ala310. Replacement of A310 by Val in the BjPutA mutant A310V raised the reduction potential of bound FAD relative to wild-type BjPutA to an E-m value of -0.09 V (pH 7.5). The > 40-mV positive shift in the potential of the BjPutA mutant A310V suggests that the corresponding Val residue in EcPutA helps poise the FAD redox potential for thermodynamically favored proline reduction thereby allowing EcPutA to be efficiently regulated by proline availability. Limited proteolysis of BjPutA under reducing conditions shows FAD reduction does not influence BjPutA conformation indicating further that the redox dependent regulation observed with EcPutA may be limited to trifunctional PutA homologues.