A H2O-producing NADH oxidase from the protozoan parasite Giardia duodenalis
A H2O-producing NADH oxidase from the protozoan parasite Giardia duodenalis
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DOI：
10.1111/j.1432-1033.1996.0155t.x
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发表时间：
1996-10-01
期刊：
影响因子：
--
通讯作者：
Upcroft, P
中科院分区：
文献类型：
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作者：
Brown, DM;Upcroft, JA;Upcroft, P
我们描述了纯化的H2O生产NADH氧化酶从原生动物寄生虫贾第鞭毛虫。该酶是一种单体黄素蛋白，含有与多肽摩尔比为1：1的黄素腺嘌呤二核苷酸。NADH氧化酶的表观分子量为46 kDa，并通过变性凝胶电泳和N-末端氨基酸测序确定是同质的。NADPH可代替NADH作为电子供体，其Km值对NADH为4.2 μ M，对NADPH为16 μ M（pH7.8）。在有氧条件下，以氧为主要电子受体，纯酶不产生O-2（.-）也没有H2 O2作为氧还原的化学计量产物，暗示H2O作为最终产物，并且避免了对超氧化物歧化酶的需要。利用氧气的能力解释了贾第虫无线粒体发酵代谢的明显呼吸作用。汞、黄酮拮抗剂和重金属（Cu 2+和Zn 2+）抑制这种活性。在厌氧条件下，该酶催化电子转移效率较低的其他电子受体，包括氮蓝四唑，铁氰化钾，FAD和FMN，使用NADH或NADPH作为电子供体。NADPH是一种更有效的电子供体。在使用的任何试验条件下，细胞色素c均未还原。该酶还原硝基呋喃类药物，呋喃唑酮（antigillardial）和呋喃妥因，其毒性自由基的形式确定的EPR。甲硝唑，硝基咪唑，没有减少。纯的NADH氧化酶没有表现出铁氧还蛋白：NAD（P）（+）氧化还原酶活性，因为它不能从还原的铁氧还蛋白接受电子以再生NAD（P）H。共和党因此，十二指肠NADH氧化酶可能作为末端氧化酶发挥作用，类似于线粒体细胞色素氧化酶，并维持最佳细胞内氧化还原比。这份关于贾第鞭毛虫黄素酶的报告将贾第鞭毛虫与厌氧菌在进化上联系在一起。
We describe the purification of a H2O-producing NADH oxidase from the protozoan parasite Giardia duodenalis. The enzyme is a monomeric flavoprotein containing flavin adenine dinucleotide in a 1:1 molar ratio with the polypeptide. The NADH oxidase has an apparent molecular mass of 46 kDa and was homogenous as determined by denaturing gel electrophoresis and N-terminal amino acid sequencing. NADPH could substitute for NADH as an electron donor with a K-m value of 4.2 mu M for NADH and 16 mu M for NADPH (pH 7.8 at room temperature). With oxygen as the primary electron acceptor under aerobic conditions, the pure enzyme did not produce O-2(.-) nor H2O2 as stoichiometric products of oxygen reduction, implicating H2O as the end product and obviating the need for superoxide dismutase. The ability to utilise oxygen explains the apparent respiration of the amitochondrial fermentative metabolism of Giardia. Mercurials, flavoantagonists and heavy metals (Cu2+ and Zn2+) inhibited this activity. Under anaerobic conditions the enzyme catalysed electron transfer at lower efficiencies to other electron accepters including nitroblue tetrazolium, potassium ferricyanide, FAD and FMN, using either NADH or NADPH as electron donors. NADPH, however, was a more efficient electron donor. Cytochrome c was not reduced under any assay conditions used. The enzyme reduced the nitrofuran drugs, furazolidone (an antigiardial) and nitrofurantoin, to their toxic radical forms as determined by EPR. Metronidazole, a nitroimidazole, was not reduced. Pure NADH oxidase did not demonstrate ferredoxin:NAD(P)(+) oxido-reductase activity since it could not accept electrons from reduced ferredoxin to regenerate NAD(P)H. The G. duodenalis NADH oxidase may, therefore, function as a terminal oxidase, similar to the mitochondrial cytochrome oxidase, and in the maintenance of an optimum intracellular redox ratio. This report of a flavoenzyme from Giardia places Giardia close to the anaerobic bacteria in evolutionary terms.