Sulfur metabolism in archaea reveals novel processes.

Studies on sulfur metabolism in archaea have revealed many novel enzymes and pathways and have advanced our understanding on metabolic processes, not only of the archaea, but of biology in general. A variety of dissimilatory sulfur metabolisms, i.e. reactions used for energy conservation, are found in archaea from both the Crenarchaeota and Euryarchaeota phyla. Although not yet fully characterized, major processes include aerobic elemental sulfur (S(0)) oxidation, anaerobic S(0) reduction, anaerobic sulfate/sulfite reduction and anaerobic respiration of organic sulfur. Assimilatory sulfur metabolism, i.e. reactions used for biosynthesis of sulfur-containing compounds, also possesses some novel features. Cysteine biosynthesis in some archaea uses a unique tRNA-dependent pathway. Fe-S cluster biogenesis in many archaea differs from that in bacteria and eukaryotes and requires unidentified components. The eukaryotic ubiquitin system is conserved in archaea and involved in both protein degradation and biosynthesis of sulfur-containing cofactors. Lastly, specific pathways are utilized for the biosynthesis of coenzyme M and coenzyme B, the sulfur-containing cofactors required for methanogenesis.

Methanogens: a window into ancient sulfur metabolism.

Methanogenesis is an ancient metabolism that originated on the early anoxic Earth. The buildup of O(2) about 2.4 billion years ago led to formation of a large oceanic sulfate pool, the onset of widespread sulfate reduction and the marginalization of methanogens to anoxic and sulfate-poor niches. Contemporary methanogens are restricted to anaerobic habitats and may have retained some metabolic relics that were common in early anaerobic life. Consistent with this hypothesis, methanogens do not utilize sulfate as a sulfur source, Cys is not utilized as a sulfur donor for Fe-S cluster and Met biosynthesis, and Cys biosynthesis uses an unusual tRNA-dependent pathway.

Biosynthesis of the salinosporamide A polyketide synthase substrate chloroethylmalonyl-coenzyme A from S-adenosyl-L-methionine.

Polyketides are among the major classes of bioactive natural products used to treat microbial infections, cancer, and other diseases. Here we describe a pathway to chloroethylmalonyl-CoA as a polyketide synthase building block in the biosynthesis of salinosporamide A, a marine microbial metabolite whose chlorine atom is crucial for potent proteasome inhibition and anticancer activity. S-adenosyl-L-methionine (SAM) is converted to 5'-chloro-5'-deoxyadenosine (5'-ClDA) in a reaction catalyzed by a SAM-dependent chlorinase as previously reported. By using a combination of gene deletions, biochemical analyses, and chemical complementation experiments with putative intermediates, we now provide evidence that 5'-ClDA is converted to chloroethylmalonyl-CoA in a 7-step route via the penultimate intermediate 4-chlorocrotonyl-CoA. Because halogenation often increases the bioactivity of drugs, the availability of a halogenated polyketide building block may be useful in molecular engineering approaches toward polyketide scaffolds.

Engineering algae for biohydrogen and biofuel production.

There is currently substantial interest in utilizing eukaryotic algae for the renewable production of several bioenergy carriers, including starches for alcohols, lipids for diesel fuel surrogates, and H2 for fuel cells. Relative to terrestrial biofuel feedstocks, algae can convert solar energy into fuels at higher photosynthetic efficiencies, and can thrive in salt water systems. Recently, there has been considerable progress in identifying relevant bioenergy genes and pathways in microalgae, and powerful genetic techniques have been developed to engineer some strains via the targeted disruption of endogenous genes and/or transgene expression. Collectively, the progress that has been realized in these areas is rapidly advancing our ability to genetically optimize the production of targeted biofuels.

Biosynthetic convergence of salinosporamides A and B in the marine actinomycete Salinispora tropica.

[structure: see text] Feeding experiments with stable isotopes established that the potent 20S-proteasome inhibitors salinosporamide A and B are biosynthesized in the marine bacterium Salinispora tropica from three biosynthetic building blocks, namely, acetate, beta-hydroxy-2'-cyclohexenylalanine, and either butyrate or a tetrose-derived chlorinated molecule. The unexpected observation that the chlorinated four-carbon residue in salinosporamide A is derived from a different metabolic origin than the non-chlorinated four-carbon unit in salinosporamide B is suggestive of a convergent biosynthesis to these two anticancer natural products.

Structural characterization of in vitro and in vivo intermediates on the loading module of microcystin synthetase.

The microcystin family of toxins is the most common cause of hepatotoxicity associated with water blooms of cyanobacterial genera. The biosynthetic assembly line producing the toxic cyclic peptide, microcystin, contains an adenylation-peptidyl carrier protein didomain (A-PCP) at the N-terminus of the initiator module McyG (295 kDa) that has been postulated to activate and load the starter unit phenylacetate for formation of the unusual aromatic beta-amino acid residue, Adda, before subsequent extension. Characterization of the McyG A-PCP didomain (78 kDa) using ATP-PP i exchange assays and mass spectrometry revealed that assorted phenylpropanoids are preferentially activated and loaded onto the PCP carrier domain rather than phenylacetate itself. For the first time, thioesters formed in vivo were detected directly using large molecule mass spectrometry. Additionally substrates were cleaved using a type II thioesterase for structural elucidation by small molecule mass spectrometry. Unprecedented features of the McyG A-PCP didomain include the in vivo acylation of the holo PCP with exogenous and endogenous substrates, along with the ability of the apo protein to retain the acyl-AMP intermediate during affinity purification. These results imply that phenylpropanoids are preferentially loaded onto the McyG PCP; however one carbon must be excised following extension of the starter unit with malonyl-CoA in order to generate the expected polyketide chain which leads us to ponder the novel biochemistry by which this occurs.