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Pyrococcus furiosus has two types of NiFe-hydrogenases: a heterotetrameric soluble hydrogenase and a multimeric transmembrane hydrogenase. Originally, the soluble hydrogenase was proposed to be a new type of H 2 evolution hydrogenase, because, in contrast to all of the then known NiFe-hydrogenases, the hydrogen production activity at 80°C was found to be higher than the hydrogen consumption activity and CO inhibition appeared to be absent. NADPH was proposed to be the electron donor. Later, it was found that the membranebound hydrogenase exhibits very high hydrogen production activity sufficient to explain cellular H 2 production levels, and this seems to eliminate the need for a soluble hydrogen production activity and therefore leave the soluble hydrogenase without a physiological function. Therefore, the steady-state kinetics of the soluble hydrogenase were reinvestigated. In contrast to previous reports, a low K m for H 2 (ϳ20 M) was found, which suggests a relatively high affinity for hydrogen. Also, the hydrogen consumption activity was 1 order of magnitude higher than the hydrogen production activity, and CO inhibition was significant (50% inhibition with 20 M dissolved CO). Since the K m for NADP ؉ is ϳ37 M, we concluded that the soluble hydrogenase from P. furiosus is likely to function in the regeneration of NADPH and thus reuses the hydrogen produced by the membrane-bound hydrogenase in proton respiration.Hydrogenase catalyzes the reversible conversion of molecular hydrogen into two protons and two electrons (8). Although hydrogenases occur in a wide variety of microorganisms from each of the three domains of life, they all have a structural blueprint in common: the active site encompasses an organometallic cofactor based on a sulfur-bridged (Fe, Fe) or (Fe, Ni) pair of metal ions with CO and CN Ϫ ligands on the Fe, with hydrophobic and hydrophilic channels to the protein's surface for transport of H 2 and protons, and with a [4Fe-4S] cubane for electron transfer (the only exception is the iron-sulfur clusterfree hydrogenase from methanogenic archaea [9]). In FeFehydrogenases the cubane is an integral part of the active site via a bridging cysteine sulfur. In NiFe-hydrogenases the cubane is in a separate subunit; however, it is spatially close to the dinuclear center, and it is thus designated a "proximal" cluster. Structures for electron transfer beyond this cubane exhibit considerable variation in hydrogenases having different origins, reflecting different functions in the metabolism of different species (6,20). Pyrococcus furiosus is a strict anaerobe and a hyperthermophilic marine archaeon that grows optimally at 100°C by saccharolytic fermentation or by S 0 -dependent peptidolytic fermentation (3). P. furiosus has two types of hydrogenases: a coenzyme-dependent heterotetrameric soluble enzyme and a multimeric (14-subunit) transmembrane enzyme. Both hydrogenases are encoded polycistronically. A duplication of the operon for the soluble enzyme encodes a lowactivity paralog, designated soluble hydro...

The physiological significance of the generic reaction H(2)<-->2[H] is not always clear because hydrogenases may function in the breakdown of molecular hydrogen or in its synthesis or in both directions. Fe-hydrogenases have nevertheless been most often associated with proton reduction and NiFe-hydrogenases with hydrogen oxidation. A re-determination of the K(M) of H(2) oxidation by Pyrococcus furiosus NiFe-hydrogenase-I and by Desulfovibrio vulgaris Fe-hydrogenase suggests that affinity for hydrogen has been seriously underestimated and that the kinetics of hydrogen activation in relation to the directionality of hydrogenases should be re-evaluated.

The exopolysaccharide produced by Streptococcus thermophilus 8S in reconstituted skimmed milk is a heteropolysaccharide containing D-galactose, D-glucose, D-ribose, and N-acetyl-D-galactosamine in a molar ratio of 2 : 1 : 1 : 1.Furthermore, the polysaccharide contains one equivalent of a novel open chain nononic acid constituent, 3,9-dideoxy-Dthreo-D-altro-nononic acid, ether-linked via C-2 to C-6 of an additional D-glucose per repeating unit. Methylation analysis and 1D/2D NMR studies ( 1 H and 13 C) performed on the native polysaccharide, and mass spectrometric and NMR analyses of the oligosaccharide obtained from the polysaccharide by de-N-acetylation followed by deamination and reduction demonstrated the ÔheptaÕsaccharide repeating unit to be:Keywords: exopolysaccharide; lactic acid bacteria; nononic acid; Streptococcus thermophilus; structural analysis.Microbial exopolysaccharides (EPSs) are employed in the food industry as viscosifying, stabilizing, emulsifying and gelling agents [1]. The texturizing properties of EPSs in fermented dairy products [2] in combination with the GRAS (generally recognized as safe) status of EPS-producing lactic acid bacteria, make these EPSs of interest for the food industry. To understand the relationship between the structure of EPSs and their physical properties, structural studies have been performed on EPSs produced by various species of the Lactobacillus, Lactococcus, and Streptococcus genera ([3,4], and references cited therein).The lactic acid bacterium Streptococcus thermophilus is used in combination with other lactic acid bacteria like Lactobacillus delbrueckii ssp. bulgaricus as starter culture for fermentations in dairy industry. In the last decade, the primary structure of the EPSs secreted by seven S. thermophilus strains [5][6][7][8][9] were elucidated. A number of the EPSs are structurally related polysaccharides and include the EPSs produced by S. thermophilus Sfi12 Recently [10], we reported for the EPS produced by S. thermophilus 8S the occurrence of a Glc residue etherified at O-6 with a novel open chain nononic acid, i.e. 6-O-(3¢,9¢-dideoxy-D-threo-D-altro-nononic acid-2¢-yl)-D-glucopyranose. Here, we report the complete structure of this EPS.

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