Abstract
Thermococcus litoralis is a strictly anaerobic archaeon (archaebacterium) that grows at temperatures up to 98 degrees C by fermenting peptides. Its growth is stimulated by tungsten, and a tungsten-containing iron-sulfur protein that has formaldehyde ferredoxin oxidoreductase (FOR) activity has been purified. FOR is a homotetramer with a subunit M(r) of 70,000. It contains approximately four irons, four acid-labile sulfides, and one tungsten atom per subunit. The tungsten appears to be present as a pterin cofactor, and the Fe/S seems to comprise an unusual [4Fe-4S] cluster that in the reduced state exists in a pH-independent S = 3/2 form and a pH-dependent S = 1/2 form. FOR catalyzed the oxidation of C1-C3 aldehydes with a temperature optimum > or = 90 degrees C and used T. litoralis ferredoxin as an electron acceptor. It did not oxidize aldehyde phosphates, utilize CoASH, or reduce NAD(P). The N-terminal sequence of FOR shows homology with the tungsto-iron-sulfur aldehyde ferredoxin oxidoreductase previously purified from the saccharolytic, hyperthermophilic archaeon Pyrococcus furiosus, in which it is proposed to function in a novel pyroglycolytic pathway (Mukund, S., and Adams, M. W. W. (1991) J. Biol. Chem. 266, 14208-14216). We show here that P. furiosus, which will also grow on peptides, albeit poorly, contains a second aldehyde-oxidizing enzyme analogous to FOR. Similarly, T. litoralis, which utilizes saccharides if limited for peptides, contains low concentrations of an enzyme analogous to AOR. It is proposed that formaldehyde (apparent Km, 62 mM) is not the true substrate for FOR; rather, the enzyme has an as yet unknown role in peptide fermentation in hyperthermophilic archaea.
Highlights
Thermococcus litoralis is a strictlyanaerobic ar- enzymes, and the organisms are considered to be the earliest chaeon that grows at temperatures known ancestors of all extant life, having evolved when the up to 98 “Cby fermentingpeptides
We show here thaPt .furiosus, which will grow on peptides, albeit poorly, contains a second aldehyde-oxidizing enzyme analogous to ferredoxin oxidoreductase (FOR)
(5mM) in 100 mM EPPS buffer, pH 8.4, at 80 "cin serum-stoppered cuvettes under argon (7, 20A).bsorbance changes were measured at we describe the properties of a second type of tungsten- 600 nm on aDMS 200 Spectrophotometer (Varian Associates) containing enzyme from a hyperthermophile, a formaldehyde ferredoxin oxidoreductase (FOR) from Therrnococcus litoralis
Summary
(Received for publication, December 14, 1992,and in revised form, March 3, 1993). From the Departmentof Biochemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602. Terminal sequence of FOR shows homology with the furwsusobtains energy for growth by the fermentation of tungsto-iron-sulfur aldehyde ferredoxin oxidoreduc- both complex and simple carbohydrates producing Hz, COZ, tase previously purified from the saccharolytic, hy- and acetate as major products It will reduce Soto H’S. perthermophilicarchaeon Pyrococcus furiosus, in Recent results suggest that So reduction is an energywhich it isproposed to function ina novel pyroglycol- conserving reaction, it does not appear to occur by yticpathway The recent discovery of microorganisms that grow optimally at temperatures near and even above 100 “C is having important ramifications in microbial physiology and metabolism, evolution, and biotechnology [1,2,3,4] They arepotential sources of a range of extraordinarilystable sequently purified from P. furiosus [19], andwas shown to be an aldehyde ferredoxin oxidoreductase (or AOR3; 20).
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