Sekine Kohsuke, Moriyama Takashi, Kim JuYaen, Hase Toshiharu, Sato Naoki
Komaba Organization for Educational Excellence, College of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan.
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153-8902, Japan.
J Biochem. 2017 Jul 1;162(1):37-43. doi: 10.1093/jb/mvw103.
Assimilatory sulfite reductase (SiR) and nitrite reductase (NiR), which are important determinants in biomass productivity, are homologous enzymes that catalyze the reduction of sulfite to sulfide and nitrite to ammonium, respectively. They have a siroheme and a [4Fe-4S] cluster as prosthetic groups in common. The red alga Cyanidioschyzon merolae encodes two SiR-like enzymes, CmSiRA and CmSiRB, which are likely products of recent gene duplication, but no homologues of NiR. The growth in a medium containing nitrate, however, must be supported by a nitrite reducing activity. CmSiRB was not detected in the ammonium medium, but, in the nitrate medium, it was present at a level 1/6 of that of constitutively expressed CmSiRA. Kinetic analysis of the two enzymes showed that CmSiRA has high kcat values with both sulfite and nitrite, but CmSiRB has virtually only the activity of nitrite reduction, although the Km value against nitrite was fairly high in both enzymes. The six amino acid residues that are specific to CmSiRB among various SiR-like enzymes in the active site were mutagenized to mimic partially CmSiRA. Among them, the mutation S217C in CmSiRB partially recovered sulfite reduction activity, suggesting that this residue is a major determinant of substrate specificity.
同化型亚硫酸盐还原酶（SiR）和亚硝酸还原酶（NiR）是生物量生产力的重要决定因素，它们是同源酶，分别催化亚硫酸盐还原为硫化物以及亚硝酸还原为铵。它们都含有一个西罗血红素和一个[4Fe-4S]簇作为共同的辅基。红藻梅氏嗜热栖热菌编码两种类SiR酶，即CmSiRA和CmSiRB，它们可能是近期基因复制的产物，但没有NiR的同源物。然而，在含有硝酸盐的培养基中生长必须有亚硝酸还原活性的支持。在铵培养基中未检测到CmSiRB，但在硝酸盐培养基中，其含量为组成型表达的CmSiRA的1/6。对这两种酶的动力学分析表明，CmSiRA对亚硫酸盐和亚硝酸盐都有较高的kcat值，但CmSiRB实际上只有亚硝酸还原活性，尽管两种酶对亚硝酸盐的Km值都相当高。在活性位点的各种类SiR酶中，CmSiRB特有的六个氨基酸残基被诱变以部分模拟CmSiRA。其中，CmSiRB中的S217C突变部分恢复了亚硫酸盐还原活性，表明该残基是底物特异性的主要决定因素。
