Biochemical evidence that the pduS gene encodes a bifunctional cobalamin reductase.
Biochemical evidence that the pduS gene encodes a bifunctional cobalamin reductase.
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生化证据表明 pduS 基因编码双功能钴胺素还原酶。
DOI：
10.1099/mic.0.27755-0
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Bobik,ThomasA
中科院分区：
文献类型：
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作者：
Sampson,EdithM;Johnson,CelesteLV;Bobik,ThomasA
肠沙门氏菌通过需要辅酶B12（腺苷钴胺素；AdoCbl）的途径降解1,2-丙二醇（1,2- pd）。专门参与1,2- pd利用（pdu）的基因是在一个大的连续簇中发现的，即pdulocus。早期的研究表明，该基因座包括将维生素B12（氰钴胺素；CNCbl）转化为AdoCbl的基因，并且pduogene编码ATP: cob(I)alamin腺苷转移酶，该酶催化该过程的最后一步。在这里，体外证据表明，pdusgene编码双功能钴胺素还原酶，催化CNCbl转化为AdoCbl所需的两个还原步骤。PduS酶在大肠杆菌中大量产生。酶分析表明，PduS表达菌株的细胞提取物将cob(III)alamin（羟钴胺素）还原为cob(II)alamin的速率为91 nmol min - 1mg - 1，将cob(II)alamin还原为cob(I)alamin的速率为7.8 nmol min - 1mg - 1。相比之下，对照提取物只有9.9 nmol min - 1mg - 1cob(III)alamin还原酶活性，没有检测到cob(II)alamin还原酶活性。因此，这些结果表明PduS酶是一种双功能钴胺素还原酶。酶分析还表明，pdu酶将cob(II)alamin还原为cob(I)alamin，并通过纯化的PduO腺苷转移酶转化为AdoCbl。此外，利用碘乙酸盐作为cob(I)alamin的化学捕集剂的研究表明，ppdu和PduO酶之间存在物理相互作用，在cob(II)alamin转化为AdoCbl的过程中，cob(I)alamin被这两种酶隔离。这在生理上可能很重要，因为cob(I)alamin具有极强的反应性，需要保护其免受非生产性副反应的影响。最后，生物信息学分析表明，PduS酶的氨基酸序列与GenBank中目前已知功能的酶无关。因此，结果表明PduS酶代表了一类新的钴胺素还原酶。
Salmonella entericadegrades 1,2-propanediol (1,2-PD) by a pathway that requires coenzyme B12(adenosylcobalamin; AdoCbl). The genes specifically involved in 1,2-PD utilization (pdu) are found in a large contiguous cluster, thepdulocus. Earlier studies have indicated that this locus includes genes for the conversion of vitamin B12(cyanocobalamin; CNCbl) to AdoCbl and that thepduOgene encodes an ATP : cob(I)alamin adenosyltransferase which catalyses the terminal step of this process. Here,in vitroevidence is presented that thepduSgene encodes a bifunctional cobalamin reductase that catalyses two reductive steps needed for the conversion of CNCbl into AdoCbl. The PduS enzyme was produced in high levels inEscherichia coli. Enzyme assays showed that cell extracts from the PduS expression strain reduced cob(III)alamin (hydroxycobalamin) to cob(II)alamin at a rate of 91 nmol min−1mg−1and cob(II)alamin to cob(I)alamin at a rate of 7·8 nmol min−1mg−1. In contrast, control extracts had only 9·9 nmol min−1mg−1cob(III)alamin reductase activity and no detectable cob(II)alamin reductase activity. Thus, these results indicated that the PduS enzyme is a bifunctional cobalamin reductase. Enzyme assays also showed that the PduS enzyme reduced cob(II)alamin to cob(I)alamin for conversion into AdoCbl by purified PduO adenosyltransferase. Moreover, studies in which iodoacetate was used as a chemical trap for cob(I)alamin indicated that the PduS and PduO enzymes physically interact and that cob(I)alamin is sequestered during the conversion of cob(II)alamin to AdoCbl by these two enzymes. This is likely to be important physiologically, since cob(I)alamin is extremely reactive and would need to be protected from unproductive by-reactions. Lastly, bioinformatic analyses showed that the PduS enzyme is unrelated in amino acid sequence to enzymes of known function currently present in GenBank. Hence, results indicate that the PduS enzyme represents a new class of cobalamin reductase.