ABSTRACT
Archaeoglobus fulgidus, a hyperthermophilic sulfate-reducing Archaeon, contains high Fe(3+)-EDTA reductase activity in its soluble protein fraction. The corresponding enzyme, which constitutes about 0.75% of the soluble protein, was purified 175-fold to homogeneity. Based on SDS-polyacrylamide gel electrophoresis, the ferric reductase consists of a single subunit with a M(r) of 18,000. The M(r) of the native enzyme was determined by size exclusion chromatography to be 40,000 suggesting that the native ferric reductase is a homodimer. The enzyme uses both NADH and NADPH as electron donors to reduce Fe(3+)-EDTA. Other Fe(3+) complexes and dichlorophenolindophenol serve as alternative electron acceptors, but uncomplexed Fe(3+) is not utilized. The purified enzyme strictly requires FMN or FAD as a catalytic intermediate for Fe(3+) reduction. Ferric reductase also reduces FMN and FAD, but not riboflavin, with NAD(P)H which classifies the enzyme as a NAD(P)H:flavin oxidoreductase. The enzyme exhibits a temperature optimum of 88 degrees C. When incubated at 85 degrees C, the enzyme activity half-life was 2 h. N-terminal sequence analysis of the purified ferric reductase resulted in the identification of the hypothetical gene, AF0830, of the A. fulgidus genomic sequence. The A. fulgidus ferric reductase shares amino acid sequence similarity with a family of NAD(P)H:FMN oxidoreductases but not with any ferric reductases suggesting that the A. fulgidus ferric reductase is a novel enzyme.
Subject(s)
Archaeal Proteins/metabolism , Archaeoglobus fulgidus/enzymology , FMN Reductase , NADH, NADPH Oxidoreductases/metabolism , Archaeal Proteins/analysis , Archaeal Proteins/genetics , Archaeal Proteins/isolation & purification , Electron Transport , Molecular Sequence Data , NADH, NADPH Oxidoreductases/analysis , NADH, NADPH Oxidoreductases/genetics , NADH, NADPH Oxidoreductases/isolation & purification , Sequence Alignment , Sequence AnalysisABSTRACT
Prenatal diagnosis of dystrophic epidermolysis bullosa (DEB) has been achieved in the past by fetal skin sampling. However, this invasive procedure is associated with a relatively high rate of pregnancy loss. We present a consanguineous Arab family ascertained by 2 affected offspring to be at risk for DEB. In a previous gestation, fetoscopic skin sampling for prenatal diagnosis yielded a false-positive result. In the index pregnancy, abnormally elevated amniotic fluid alpha-fetoprotein (13.7 MOM) and positive acetylcholinesterase were highly suggestive of an affected fetus. Fetal skin biopsy was declined. At term, the patient delivered a male infant with DEB that expired on the 3rd day of life. It is apparent from our experience and from review of the literature that in some genodermatoses, markedly elevated alpha-fetoprotein and positive acetylcholinesterase in amniotic fluid are highly suggestive of an affected fetus and may obviate the need for fetal skin sampling in the prenatal diagnosis of these disorders.