Bergmann D J, Zahn J A, Hooper A B, DiSpirito A A
Department of Microbiology, Iowa State University, Ames, Iowa 50011, USA.
J Bacteriol. 1998 Dec;180(24):6440-5. doi: 10.1128/JB.180.24.6440-6445.1998.
P460 cytochromes catalyze the oxidation of hydroxylamine to nitrite. They have been isolated from the ammonia-oxidizing bacterium Nitrosomonas europaea (R. H. Erickson and A. B. Hooper, Biochim. Biophys. Acta 275:231-244, 1972) and the methane-oxidizing bacterium Methylococcus capsulatus Bath (J. A. Zahn et al., J. Bacteriol. 176:5879-5887, 1994). A degenerate oligonucleotide probe was synthesized based on the N-terminal amino acid sequence of cytochrome P460 and used to identify a DNA fragment from M. capsulatus Bath that contains cyp, the gene encoding cytochrome P460. cyp is part of a gene cluster that contains three open reading frames (ORFs), the first predicted to encode a 59,000-Da membrane-bound polypeptide, the second predicted to encode a 12, 000-Da periplasmic protein, and the third (cyp) encoding cytochrome P460. The products of the first two ORFs have no apparent similarity to any proteins in the GenBank database. The overall sequence similarity of the P460 cytochromes from M. capsulatus Bath and N. europaea was low (24.3% of residues identical), although short regions of conserved residues are present in the two proteins. Both cytochromes have a C-terminal, c-heme binding motif (CXXCH) and a conserved lysine residue (K61) that may provide an additional covalent cross-link to the heme (D. M. Arciero and A. B. Hooper, FEBS Lett. 410:457-460, 1997). Gene probing using cyp indicated that a cytochrome P460 similar to that from M. capsulatus Bath may be present in the type II methanotrophs Methylosinus trichosporium OB3b and Methylocystis parvus OBBP but not in the type I methanotrophs Methylobacter marinus A45, Methylomicrobium albus BG8, and Methylomonas sp. strains MN and MM2. Immunoblot analysis with antibodies against cytochrome P460 from M. capsulatus Bath indicated that the expression level of cytochrome P460 was not affected either by expression of the two different methane monooxygenases or by addition of ammonia to the culture medium.
P460 细胞色素催化羟胺氧化为亚硝酸盐。它们已从氨氧化细菌欧洲亚硝化单胞菌（R. H. 埃里克森和 A. B. 胡珀，《生物化学与生物物理学报》275:231 - 244，1972 年）和甲烷氧化细菌荚膜甲基球菌巴斯菌株（J. A. 扎恩等人，《细菌学杂志》176:5879 - 5887，1994 年）中分离出来。基于细胞色素 P460 的 N 端氨基酸序列合成了简并寡核苷酸探针，并用于鉴定来自荚膜甲基球菌巴斯菌株的一个 DNA 片段，该片段包含编码细胞色素 P460 的基因 cyp。cyp 是一个基因簇的一部分，该基因簇包含三个开放阅读框（ORF），第一个预计编码一个 59,000 道尔顿的膜结合多肽，第二个预计编码一个 12,000 道尔顿的周质蛋白，第三个（cyp）编码细胞色素 P460。前两个 ORF 的产物与 GenBank 数据库中的任何蛋白质均无明显相似性。来自荚膜甲基球菌巴斯菌株和欧洲亚硝化单胞菌的 P460 细胞色素的整体序列相似性较低（24.3%的残基相同），尽管这两种蛋白质中存在保守残基的短区域。两种细胞色素都有一个 C 端的 c - 血红素结合基序（CXXCH）和一个保守的赖氨酸残基（K61），该赖氨酸残基可能为血红素提供额外的共价交联（D. M. 阿奇埃罗和 A. B. 胡珀，《欧洲生物化学学会联合会快报》410:457 - 460，1997 年）。使用 cyp 进行基因探测表明，与来自荚膜甲基球菌巴斯菌株的细胞色素 P460 相似的细胞色素 P460 可能存在于 II 型甲烷氧化菌 Trichosporium 甲基弯曲菌 OB3b 和微小甲基孢囊菌 OBBP 中，但不存在于 I 型甲烷氧化菌海生甲基杆菌 A45、白色甲基微菌 BG8 和甲基单胞菌属菌株 MN 和 MM2 中。用针对荚膜甲基球菌巴斯菌株细胞色素 P460 的抗体进行免疫印迹分析表明，细胞色素 P460 的表达水平不受两种不同甲烷单加氧酶表达的影响，也不受向培养基中添加氨的影响。
