In methanogenic archaea, the carbon dioxide (CO) fixation and methane-forming steps are linked through the heterodisulfide reductase (HdrABC)-[NiFe]-hydrogenase (MvhAGD) complex that uses flavin-based electron bifurcation to reduce ferredoxin and the heterodisulfide of coenzymes M and B. Here, we present the structure of the native heterododecameric HdrABC-MvhAGD complex at 2.15-angstrom resolution. HdrB contains two noncubane [4Fe-4S] clusters composed of fused [3Fe-4S]-[2Fe-2S] units sharing 1 iron (Fe) and 1 sulfur (S), which were coordinated at the CCG motifs. Soaking experiments showed that the heterodisulfide is clamped between the two noncubane [4Fe-4S] clusters and homolytically cleaved, forming coenzyme M and B bound to each iron. Coenzymes are consecutively released upon one-by-one electron transfer. The HdrABC-MvhAGD atomic model serves as a structural template for numerous HdrABC homologs involved in diverse microbial metabolic pathways.

Human acid ceramidase ((AC) N-acylsphingosine amidohydrolase, EC 3.5.1.23) hydrolyzes the sphingolipid ceramide into sphingosine and free fatty acid. Ceramide is an essential component of all sphingolipids and an important cell-signaling molecule. Moreover, an inherited deficiency of AC activity leads to the lysosomal storage disorder known as Farber disease. Human AC was purified from urine, and 117 amino acid residues were determined by microsequencing. Degenerative oligonucleotide probes were then constructed and used to screen for human fibroblast and pituitary cDNA libraries. Several partial cDNA clones were obtained, and two of these were combined to construct a full-length cDNA containing a 17-base pair (bp) 5-untranslated sequence, a 1185-bp open reading frame encoding 395 amino acids, a 1110-bp 3-untranslated sequence, and an 18-bp poly(A) tail. Transient expression of the full-length cDNA in COS-1 cells led to a 10-fold increase in AC activity. In addition, biosynthetic studies carried out in the transfected cells demonstrated that 13-kDa (␣) and 40-kDa (␤) AC subunits were derived from a common 55-kDa precursor encoded by the full-length cDNA. This protein pattern was identical to that seen in normal human skin fibroblasts. A homoallelic point mutation (T222K) was also identified in the AC gene of a patient suffering from Farber disease, further confirming the authenticity of the full-length cDNA.Human acid ceramidase ((AC) 1 N-acylsphingosine amidohydrolase, EC 3.5.1.23) catalyzes the hydrolysis of ceramide to free fatty acid and sphingosine (1). An inherited deficiency of AC activity leads to the lysosomal storage disorder known as Farber disease (FD), also called Farber lipogranulomatosis (2). Patients with FD accumulate ceramide in most tissues, leading to painful swelling of the joints and tendons, pulmonary insufficiency, and a shortened life-span. The clinical diagnosis of FD is usually confirmed by biochemical methods, including the determination of lysosomal ceramide accumulation and/or the deficiency of AC activity. To date, seven FD subtypes have been described with varying degrees of clinical involvement; notably, a direct correlation between the amount of ceramide accumulation and the clinical severity of FD patients has recently been demonstrated (3).In addition to its central role in disease pathogenesis, sphingolipid biosynthesis, and membrane formation, ceramide is an important cell-signaling molecule involved in a variety of diverse processes such as neurite growth, monocyte differentiation, and Fas (Apo1/CD95)-induced apoptosis (for reviews, see Refs. 4 and 5). Moreover, sphingosine (the catabolic product of ceramide degradation) has been shown to inhibit protein kinase C activity and can exert a variety of effects on cell growth and differentiation (6 -8). Since ceramide degradation is the only catabolic source of intracellular sphingosine (9, 10), AC activity may be the rate-limiting step in determining the intracellular levels of this compound.About 2 years ago, AC was purified...

Methanosarcina barkeri has recently been shown to produce a multisubunit membrane-bound [NiFe] hydrogenase designated Ech (Escherichia coli hydrogenase 3) hydrogenase. In the present study Ech hydrogenase was purified to apparent homogeneity in a high yield. The enzyme preparation obtained only contained the six polypeptides which had previously been shown to be encoded by the ech operon. The purified enzyme was found to contain 0.9 mol of Ni, 11.3 mol of nonheme-iron and 10.8 mol of acid-labile sulfur per mol of enzyme. Using the purified enzyme the kinetic parameters were determined. The enzyme catalyzed the H 2 dependent reduction of a M. barkeri 2[4Fe-4S] ferredoxin with a specific activity of 50 U´mg protein 21 at pH 7.0 and exhibited an apparent K m for the ferredoxin of 1 mm. The enzyme also catalyzed hydrogen formation with the reduced ferredoxin as electron donor at a rate of 90 U´mg protein 21 at pH 7.0. The apparent K m for the reduced ferredoxin was 7.5 mm. Reduction or oxidation of the ferredoxin proceeded at similar rates as the reduction or oxidation of oxidized or reduced methylviologen, respectively. The apparent K m for H 2 was 5 mm. The kinetic data strongly indicate that the ferredoxin is the physiological electron donor or acceptor of Ech hydrogenase. Ech hydrogenase amounts to about 3% of the total cell protein in acetate-grown, methanol-grown or H 2 /CO 2 -grown cells of M. barkeri, as calculated from quantitative Western blot experiments. The function of Ech hydrogenase is ascribed to ferredoxin-linked H 2 production coupled to the oxidation of the carbonyl-group of acetyl-CoA to CO 2 during growth on acetate, and to ferredoxin-linked H 2 uptake coupled to the reduction of CO 2 to the redox state of CO during growth on H 2 /CO 2 or methanol.Keywords: Methanosarcina barkeri; hydrogenase; complex I; energy conservation; iron±sulfur protein; ferredoxin.Methanosarcina barkeri is a methanogenic archaeon that can utilize H 2 /CO 2 , methanol or methylamines as energy substrates (reviewed in [1±3]). Three different [NiFe] hydrogenases have been characterized from Methanosarcina species: F 420 -reducing hydrogenase, F 420 -nonreducing hydrogenase and Ech hydrogenase.F 420 -reducing hydrogenase has been purified and characterized from M. barkeri [4,5]. The enzyme catalyzes the reduction of coenzyme F 420 which plays an important role as redox carrier in methanogenic archaea. The genome of M. barkeri contains two gene clusters ( frh and fre) encoding two related F 420 -reducing hydrogenases indicating the presence of two isoenzymes. Both operons were transcribed during growth on H 2 /CO 2 , methanol or trimethylamine [6]. In acetategrown cells no transcripts of these operons were detectable although these cells contain small amounts of F 420 -reducing hydrogenase activity (about 5% of the activity detectable in methanol-grown cells; Meuer and Hedderich, unpublished results).F 420 -nonreducing hydrogenase has been purified from M. barkeri and from Methanosarcina mazei. The purified enzyme was fo...

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Social behavior in the bacterium Myxococcus xanthus relies on contact-dependent activities involving cell-cell and cell-substratum interactions. To identify outer membrane proteins that have a role in these activities, we profiled the outer membrane proteome of growing and starving cells using two strategies. First, outer membrane proteins were enriched by biotinylation of intact cells using the reagent NHS (N-hydroxysuccinimide)-PEO(12) (polyethylene oxide)-biotin with subsequent membrane solubilization and affinity chromatography. Second, the proteome of outer membrane vesicles (OMV) was determined. Comparisons of detected proteins show that these methods have different detection profiles and together provide a comprehensive view of the outer membrane proteome. From 362 proteins identified, 274 (76%) were cell envelope proteins including 64 integral outer membrane proteins and 85 lipoproteins. The majority of these proteins were of unknown function. Among integral outer membrane proteins with homologues of known function, TonB-dependent transporters comprise the largest group. Our data suggest novel functions for these transporters. Among lipoproteins with homologues of known function, proteins with hydrolytic functions comprise the largest group. The luminal load of OMV was enriched for proteins with hydrolytic functions. Our data suggest that OMV have functions in predation and possibly in transfer of intercellular signaling molecules between cells.