Since 1933, carbonic anhydrase research has focused on enzymes from mammals (␣ class) and plants (␤ class); however, two additional classes (␥ and ␦) were discovered recently. Cam, from the procaryote Methanosarcina thermophila, is the prototype of the ␥ class and the first carbonic anhydrase to be characterized from either an anaerobic organism or the Archaea domain. All of the enzymes characterized from the four classes have been purified aerobically and are reported to contain a catalytic zinc. Herein, we report the anaerobic reconstitution of apo- Research in the intervening years has shown that CA is one of the most widely distributed enzymes in nature (2, 3) and continues to be intensely investigated. Amino acid sequence comparisons identify four classes (␣, ␤, ␥, and ␦) of independent origins (4). Isozymes of the ␣ class are found in virtually all mammalian tissues where they function in diverse essential processes. The ␤ class is ubiquitous in plants and algae, where it is indispensable for the acquisition and concentration of CO 2 for photosynthesis. CA plays a role in the sequestration of atmospheric CO 2 in carbonates, and the global cycles of silicon and carbon are linked by CA in diatoms (5); thus, CA plays an important role in major geochemical and atmospheric processes. Members of the ␤ and ␥ classes are wide spread in physiologically diverse procaryotes from both the Bacteria and Archaea domains. Indeed, the genome of Escherichia coli contains two ␥ class homologs and two ␤ class homologs (2).Cam, from the procaryote Methanosarcina thermophila, is the prototype of the ␥ class and the first CA to be characterized from either an anaerobic organism or the Archaea domain (6). Sequence analyses approximate the evolution of the ␥ class at the estimated time of the origin of life (2). The crystal structure of Cam purified aerobically from E. coli reveals a homotrimer with a subunit fold composed of a left-handed ␤-helix motif followed by short and long ␣-helix structures (7). Each of the three active sites contain three histidines that coordinate a zinc ion. Two of the metal-binding histidines are donated by one monomer, and the third histidine from an adjacent monomer. Other residues in the active site of Cam are also donated from adjacent monomer faces and bear no resemblance to residues in the active site of the well characterized ␣ class CAs for which specific functions have been assigned.Kinetic investigations of the ␣ class CAs reveal a "zinc hydroxide" mechanism for catalysis (8) that also extends to both the ␤ and ␥ classes (9, 10). The overall enzyme-catalyzed reaction occurs in two mechanistically distinct steps,where E is enzyme, and B is buffer. The first step is the interconversion between CO 2 and bicarbonate (Equations 2 and 3) involving a nucleophilic attack of the zinc-bound hydroxyl on the CO 2 molecule. The second step is regeneration of the zinc-bound hydroxide, which involves intramolecular proton transfer from the zinc-bound water to a proton shuttle residue (Equation 4) and interm...

A total of 35 homologs of the iron-sulfur flavoprotein (Isf) from Methanosarcina thermophila were identified in databases. All three domains were represented, and multiple homologs were present in several species. An unusually compact cysteine motif ligating the 4Fe-4S cluster in Isf is conserved in all of the homologs except two, in which either an aspartate or a histidine has replaced the second cysteine in the motif. A phylogenetic analysis of Isf homologs identified four subgroups, two of which were supported by bootstrap data. Three homologs from metabolically and phylogenetically diverse species in the Bacteria and Archaea domains (Af3 from Archaeoglobus fulgidus, Cd1 from Clostridium difficile, and Mj2 from Methanococcus jannaschii) were overproduced in Escherichia coli. Each homolog purified as a homodimer, and the UV-visible absorption spectra were nearly identical to that of Isf. After reconstitution with iron, sulfide, and flavin mononucleotide (FMN) the homologs contained six to eight nonheme iron atoms and 1.6 to 1.7 FMN molecules per dimer, suggesting that two 4Fe-4S or 3Fe-4S clusters and two FMN cofactors were bound to each dimer, which is consistent with Isf data. Homologs Af3 and Mj2 were reduced by CO in reactions catalyzed by cell extract of acetate-grown M. thermophila, but Cd1 was not. Homologs Af3 and Mj2 were reduced by CO in reactions catalyzed by A. fulgidus and M. jannaschii cell extracts. Cell extract of Clostridium thermoaceticum catalyzed CO reduction of Cd1. Our database sequence analyses and biochemical characterizations indicate that Isf is the prototype of a family of iron-sulfur flavoproteins that occur in members of all three domains.Methane produced by the acetate fermentation pathway accounts for two-thirds of all biologically produced methane. In Methanosarcina thermophila, acetate is cleaved into carbonyl and methyl groups by the CO dehydrogenase (CODH)-acetyl coenzyme A (acetyl-CoA) synthase (ACS) enzyme complex. The methyl group is subsequently reduced to methane by electrons derived from oxidation of the carbonyl group to CO 2 by the CODH-ACS complex (7). An iron-sulfur flavoprotein (Isf) from M. thermophila has been overproduced in Escherichia coli, purified, and characterized. Isf is purified as a homodimer which binds two 4Fe-4S clusters and two flavin mononucleotide (FMN) cofactors (2, 14). Ferredoxin, the electron acceptor of the CODH-ACS complex, is the physiological electron donor for Isf. Additional results support the hypothesis that Isf plays a role in electron transport during fermentation of acetate to methane (14). The deduced amino acid sequence of Isf contains an unusually compact cysteine motif (CX 2 CX 2 CX 5 C) that has been shown, on the basis of site-directed mutagenesis and electron paramagnetic resonance studies, to ligate the 4Fe-4S cluster (15). The midpoint potential values are -394 mV for the 4Fe-4S cluster and -277 mV for the FMN in Isf, suggesting that electrons derived from oxidation of the carbonyl group of acetate flow from ferredoxin to t...

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1-O-Hexadecylglycerol (chimyl alcohol), 1-O-heptadecylglycerol and 1-O-octadecylglycerol (batyl alcohol) have been identified as the major native constituents of a mixture of free alkyl glycerol ethers isolated from the contained water and the methanolic extract of the sponge Desmapsamma anchorata. Minor components were the free C14, C15, C19, C20 and C21 alkyl glycerol monoethers. The alkyl glycerol monoethers were analyzed and identified by gas chromatography/mass spectrometry of their isopropylidene derivatives. This is the first report on the occurrence of free C15, C19, C20 and C21 alkyl glycerol monoethers in a sponge.

Iron-sulfur flavoproteins (ISF) constitute a widespread family of redox-active proteins in anaerobic prokaryotes. Based on sequence homologies, their overall structure is expected to be similar to that of flavodoxins, but in addition to a flavin mononucleotide cofactor they also contain a cubane-type [4Fe:4S] cluster. In order to gain further insight into the function and properties of ISF, the three-dimensional structures of two ISF homologs, one from the thermophilic methanogen Methanosarcina thermophila and one from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus, were determined. The structures indicate that ISF assembles to form a tetramer and that electron transfer between the two types of redox cofactors requires oligomerization to juxtapose the flavin mononucleotide and [4Fe:4S] cluster bound to different subunits. This is only possible between different monomers upon oligomerization. Fundamental differences in the surface properties of the two ISF homologs underscore the diversity encountered within this protein family.The ISF (iron-sulfur flavoprotein) family is distinct from other flavin mononucleotide (FMN)-containing flavoproteins based on overall sequence identity and an unusually compact cysteine motif (24). The prototype of ISF was characterized from Methanosarcina thermophila (ISF-Mt), a strictly anaerobic methane-producing thermophile belonging to the domain Archaea (16). Since then, homologs of ISF have been identified in the genomes of numerous anaerobic prokaryotes belonging to the domains Bacteria and Archaea (24). Remarkably, many species contain multiple ISF homologs; indeed, the Methanosarcina acetivorans genome is annotated with 19 homologs (12). The only ISF homolog identified from a member of the domain Eukarya is that from the intestinal anaerobic pathogen Entamoeba histolytica (24). Thus, it appears that the ISF family occurs predominantly in anaerobic prokaryotes.ISF from M. thermophila has been reported to be a functional homodimer, with each monomer containing one FMN and one [4Fe:4S] cluster (1, 16). Ferredoxin can act as an electron donor, and redox potential measurements support the hypothesis that electron flow proceeds from ferredoxin to the low-potential (Ϫ394 mV) [4Fe:4S] cluster and then to the flavin (1). Although a downstream electron acceptor is unknown, the inability to detect flavin semiquinone during redox titrations suggests that the protein environment of ISF-Mt stabilizes the hydroquinone form and that ISF-Mt functions as a one-electron/two-electron switch. A role for ISF in electron transport coupled to methane formation has been postulated (16); however, the presence of multiple ISF homologs in metabolically diverse anaerobic prokaryotes (24) suggests broader physiological functions.The cysteine motif (Cys47, Cys50, Cys53, and Cys59) in ISF-Mt is highly conserved among the deduced sequences of all ISF homologs, consistent with a role in ligation of the [4Fe:4S] cluster. The compact nature of the motif is unusual compared to motifs known t...