Purification and Molecular Characterization of the Tungsten-Containing Formaldehyde Ferredoxin Oxidoreductase from the Hyperthermophilic Archaeon Pyrococcus furiosus: the Third of a Putative Five-Member Tungstoenzyme Family
Purification and Molecular Characterization of the Tungsten-Containing Formaldehyde Ferredoxin Oxidoreductase from the Hyperthermophilic Archaeon Pyrococcus furiosus: the Third of a Putative Five-Member Tungstoenzyme Family
复制标题
来自超嗜热古菌激烈火球菌的含钨甲醛铁氧还蛋白氧化还原酶的纯化和分子表征：推定的五成员钨酶家族中的第三种
作者：
R. Roy;S. Mukund;G. Schut;D. Dunn;R. Weiss;M. Adams
摘要激烈热球菌是一种极端嗜热古菌，其最佳生长温度接近100°C，通过发酵肽和糖产生有机酸、CO2和H2。它的生长需要钨，并且先前已经从P. furiosus中纯化了两种不同的含钨酶，醛铁氧还蛋白氧化还原酶（AOR）和甘油醛-3-磷酸铁氧还蛋白氧化还原酶（GAPOR）。这两种酶被认为分别在肽和碳水化合物的代谢中起作用。第三种类型的含钨酶，甲醛铁氧还蛋白氧化还原酶（FOR），现在已经确定。FOR是一个同源四聚体，分子量为280 kDa，每个亚基含有约1个W原子、4个Fe原子和1个Ca原子，以及一个蝶呤辅因子。在纯化过程中FOR活性的低回收率归因于硫化物的损失，因为在还原条件下用硫化物（HS-）处理纯化的酶被激活高达五倍。供使用。furiosus铁氧还蛋白作为电子受体（Km = 100 μM），并氧化一系列醛。在常规测定中使用甲醛（对于硫化物活化的酶，Km = 15 mM），但认为生理底物是脂族C5半醛或二醛，例如，戊二醛（Km = 1 mM）。基于其氨基末端序列，在基因组数据库中鉴定了编码FOR（for）的基因，以及编码AOR和GAPOR的基因。FOR的氨基酸序列对应于68.7 kDa的质量，并且与AOR（61%相似性和40%同一性）和GAPOR（50%相似性和23%同一性）的亚基的氨基酸序列高度相似。这三个基因在P. furiosus染色体上不连锁。两个额外的（和非连锁）基因（termedwor 4和wor 5），编码推定的钨酶与57%（WOR 4）和56%（WOR 5）的序列相似性FOR也被确定。基于与FOR的序列基序相似性，也提出了W 0 R4和W 0 R5都含有钨双蝶呤位点和每个亚基一个[4Fe-4S]簇。
ABSTRACT Pyrococcus furiosus is a hyperthermophilic archaeon which grows optimally near 100°C by fermenting peptides and sugars to produce organic acids, CO2, and H2. Its growth requires tungsten, and two different tungsten-containing enzymes, aldehyde ferredoxin oxidoreductase (AOR) and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR), have been previously purified from P. furiosus. These two enzymes are thought to function in the metabolism of peptides and carbohydrates, respectively. A third type of tungsten-containing enzyme, formaldehyde ferredoxin oxidoreductase (FOR), has now been characterized. FOR is a homotetramer with a mass of 280 kDa and contains approximately 1 W atom, 4 Fe atoms, and 1 Ca atom per subunit, together with a pterin cofactor. The low recovery of FOR activity during purification was attributed to loss of sulfide, since the purified enzyme was activated up to fivefold by treatment with sulfide (HS−) under reducing conditions. FOR usesP. furiosus ferredoxin as an electron acceptor (Km = 100 μM) and oxidizes a range of aldehydes. Formaldehyde (Km = 15 mM for the sulfide-activated enzyme) was used in routine assays, but the physiological substrate is thought to be an aliphatic C5semi- or dialdehyde, e.g., glutaric dialdehyde (Km = 1 mM). Based on its amino-terminal sequence, the gene encoding FOR (for) was identified in the genomic database, together with those encoding AOR and GAPOR. The amino acid sequence of FOR corresponded to a mass of 68.7 kDa and is highly similar to those of the subunits of AOR (61% similarity and 40% identity) and GAPOR (50% similarity and 23% identity). The three genes are not linked on the P. furiosuschromosome. Two additional (and nonlinked) genes (termedwor4 and wor5) that encode putative tungstoenzymes with 57% (WOR4) and 56% (WOR5) sequence similarity to FOR were also identified. Based on sequence motif similarities with FOR, both WOR4 and WOR5 are also proposed to contain a tungstobispterin site and one [4Fe-4S] cluster per subunit.