de Boer A P, van der Oost J, Reijnders W N, Westerhoff H V, Stouthamer A H, van Spanning R J
Department of Microbial Physiology, Faculty of Biology, BioCentrum Amsterdam, Vrije Universiteit, The Netherlands.
Eur J Biochem. 1996 Dec 15;242(3):592-600. doi: 10.1111/j.1432-1033.1996.0592r.x.
The genes that encode the hc-type nitric-oxide reductase from Paracoccus denitrificans have been identified. They are part of a cluster of six genes (norCBQDEF) and are found near the gene cluster that encodes the cd1-type nitrite reductase, which was identified earlier [de Boer, A. P. N., Reijnders, W. N. M., Kuenen, J. G., Stouthamer, A. H. & van Spanning, R. J. M. (1994) Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans, Antonie Leeu wenhoek 66, 111-127]. norC and norB encode the cytochrome-c-containing subunit II and cytochrome b-containing subunit I of nitric-oxide reductase (NO reductase), respectively. norQ encodes a protein with an ATP-binding motif and has high similarity to NirQ from Pseudomonas stutzeri and Pseudomonas aeruginosa and CbbQ from Pseudomonas hydrogenothermophila. norE encodes a protein with five putative transmembrane alpha-helices and has similarity to CoxIII, the third subunit of the aa3-type cytochrome-c oxidases. norF encodes a small protein with two putative transmembrane alpha-helices. Mutagenesis of norC, norB, norQ and norD resulted in cells unable to grow anaerobically. Nitrite reductase and NO reductase (with succinate or ascorbate as substrates) and nitrous oxide reductase (with succinate as substrate) activities were not detected in these mutant strains. Nitrite extrusion was detected in the medium, indicating that nitrate reductase was active. The norQ and norD mutant strains retained about 16% and 23% of the wild-type level of NorC, respectively. The norE and norF mutant strains had specific growth rates and NorC contents similar to those of the wild-type strain, but had reduced NOR and NIR activities, indicating that their gene products are involved in regulation of enzyme activity. Mutant strains containing the norCBQDEF region on the broad-host-range vector pEG400 were able to grow anaerobically, although at a lower specific growth rate and with lower NOR activity compared with the wild-type strain.
已鉴定出反硝化副球菌中编码hc型一氧化氮还原酶的基因。它们是一个由六个基因组成的基因簇（norCBQDEF）的一部分，位于编码cd1型亚硝酸还原酶的基因簇附近，该基因簇已于较早时候被鉴定出来[de Boer, A. P. N., Reijnders, W. N. M., Kuenen, J. G., Stouthamer, A. H. & van Spanning, R. J. M. (1994) 反硝化副球菌中亚硝酸还原相关基因簇的分离、测序及突变分析，Antonie Leeuwenhoek 66, 111 - 127]。norC和norB分别编码一氧化氮还原酶（NO还原酶）中含细胞色素c的亚基II和含细胞色素b的亚基I。norQ编码一种具有ATP结合基序的蛋白质，与施氏假单胞菌、铜绿假单胞菌中的NirQ以及嗜氢假单胞菌中的CbbQ具有高度相似性。norE编码一种具有五个推定跨膜α螺旋的蛋白质，与aa3型细胞色素c氧化酶的第三个亚基CoxIII具有相似性。norF编码一种具有两个推定跨膜α螺旋的小蛋白质。对norC、norB、norQ和norD进行诱变导致细胞无法在厌氧条件下生长。在这些突变菌株中未检测到亚硝酸还原酶和NO还原酶（以琥珀酸或抗坏血酸为底物）以及一氧化二氮还原酶（以琥珀酸为底物）的活性。在培养基中检测到亚硝酸盐外排，表明硝酸还原酶具有活性。norQ和norD突变菌株分别保留了野生型NorC水平的约16%和23%。norE和norF突变菌株的比生长速率和NorC含量与野生型菌株相似，但NOR和NIR活性降低，表明它们的基因产物参与酶活性的调节。含有广泛宿主范围载体pEG400上norCBQDEF区域的突变菌株能够在厌氧条件下生长，尽管与野生型菌株相比，其比生长速率较低且NOR活性较低。
