Archaea have been shown to produce isoprenoids from mevalonate; however, genome analysis has failed to identify several genes in the mevalonate pathway on the basis of sequence similarity. A predicted archaeal kinase, coded for by the MJ0044 gene, was associated with other mevalonate pathway genes in the archaea and was predicted to be the "missing" phosphomevalonate kinase. The MJ0044-derived protein was tested for phosphomevalonate kinase activity and was found not to catalyze this reaction. The MJ0044 gene product was found to phosphorylate isopentenyl phosphate, generating isopentenyl diphosphate. Unlike other known kinases associated with isoprene biosynthesis, Methanocaldococcus jannaschii isopentenyl phosphate kinase is predicted to be a member of the aspartokinase superfamily.Isoprenoids are a large family of natural products that includes metabolically and medically important compounds such as cholesterol, steroid hormones, ubiquinone, carotenoids, and taxol. In archaea, isoprenoids are of particular interest because they are the major component of their membrane lipids (22). Archaeal lipids are composed of isoprenoid side chains connected to sn-glycerol-1-phosphate through ether linkages. Thus, the archaeal lipids differ from the majority of eukaryotic and eubacterial lipids in the isoprene nature of the alkyl chain, the stereoconfiguration of the glycerol moiety, and the presence of ether rather than ester linkages (22).Despite the structural diversity seen in this group of compounds, all isoprenoids are derived from two precursor compounds: isopentenyl diphosphate (IPP) and its isomer 3,3-dimethylallyl diphosphate. Two pathways for the biosynthesis of these central metabolites are currently known: the mevalonate and the deoxy-D-xylulose 5-phosphate (DXP) pathways (19). The DXP pathway is known to function in the majority of bacteria and plant plastids, while the mevalonate pathway is typically found in animals, plant cytosol, and archaea. Some bacteria, along with plants, have been shown to operate with both pathways (19).Labeled precursor studies have shown that both acetate and mevalonate are precursors for isoprenoids in archaea (13,14,33). These observations were supported through archaeal genome analysis which revealed homologs of mevalonate pathway enzymes, including 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase and HMG-CoA reductase (9,20,31). Putative genes for other enzymes in the mevalonate pathway, however, were not immediately identified on the basis of sequence similarity. However, two additional enzymes of the pathway, mevalonate kinase and IPP isomerase, were subsequently identified and characterized (4, 17).Although Sulfolobus tokodaii and Sulfolobus solfataricus have homologs for all mevalonate pathway enzymes, genomic analysis was unable to identify genes for phosphomevalonate kinase or diphosphomevalonate decarboxylase in most archaea (7). Similarly, several species of Halobacteria have gene homologs for diphosphomevalonate decarboxylase but not for phosphomevalonate kin...

Blasticidin S is a potent antifungal and cytotoxic peptidyl nucleoside antibiotic from Streptomyces griseochromogenes. The mixed biosynthesis of the compound is evident from the three distinct structural components: a cytosine base, an amino deoxyglucuronic acid, and N-methyl beta-arginine. The blasticidin S biosynthesis gene cluster was cloned from S. griseochromogenes and the pathway heterologously expressed in S. lividans from a cosmid harboring a 36.7-kb fragment of S. griseochromogenes DNA. The complete DNA sequence of this insert has now been determined and evidence suggests a contiguous 20-kb section defines the blasticidin S biosynthesis cluster. The predicted functions of several open reading frames are consistent with the expected biochemistry and include an arginine 2,3-aminomutase, a cytosylglucuronic acid synthase, and a guanidino N-methyltransferase. Insight into other steps in the assembly of blasticidin S was evident from sequence homology with proteins of known function and heterologous expression of fragments of the cluster. Additionally, the gene that directs the production of free cytosine, blsM, was subcloned and expressed in Escherichia coli. Characterization of BlsM revealed that cytidine monophosphate serves as the precursor to cytosine.

Coenzyme F 420 , a hydride carrier, is found in Archaea and some bacteria and has crucial roles in methanogenesis, antibiotic biosynthesis, DNA repair, and activation of antitubercular compounds. CofD, 2-phospho-L-lactate transferase, catalyzes the last step in the biosynthesis of F 420 -0 (F 420 without polyglutamate), by transferring the lactyl phosphate moiety of lactyl(2)diphospho-(5)guanosine to 7,8-didemethyl-8-hydroxy-5-deazariboflavin ribitol (Fo). CofD is highly conserved among F 420 -producing organisms, and weak sequence homologs are also found in non-F 420 -producing organisms. This superfamily does not share any recognizable sequence conservation with other proteins. Here we report the first crystal structures of CofD, the free enzyme and two ternary complexes, with Fo and P i or with Fo and GDP, from Methanosarcina mazei. The active site is located at the C-terminal end of a Rossmann fold core, and three large insertions make significant contributions to the active site and dimer formation. The observed binding modes of Fo and GDP can explain known biochemical properties of CofD and are also supported by our binding assays. The structures provide significant molecular insights into the biosynthesis of the F 420 coenzyme. Large structural differences in the active site region of the non-F 420 -producing CofD homologs suggest that they catalyze a different biochemical reaction.The coenzyme F 420 is a hydride carrier that is found in Archaea and in high GϩC Gram-positive bacteria such as Streptomyces and Mycobacterium. This coenzyme is essential for energy metabolism in methanogenic Archaea, which involves the conversion of CO 2 , several other one-carbon compounds, and acetate to CH 4 (1-4). In Streptomyces, F 420 is involved in the biosynthesis of tetracycline, lincomycin, and other natural products (5-9). In some cyanobacteria, a precursor of F 420 is a cofactor in DNA photolyases for DNA repair (10, 11). In Mycobacterium tuberculosis, an F 420 -dependent glucose-6-phosphate dehydrogenase is required for the reductive activation of a series of nitroimidazo-oxazine compounds for their antitubercular effects (12)(13)(14).Coenzyme F 420 is named for the intense absorption at 420 nm by the oxidized form of this compound. The chromophore is 7,8-didemethyl-8-hydroxy-5-deazariboflavin, which is linked to a ribityl group at its N-10 position to produce Fo 3 (Fig. 1A). Fo is covalently linked to a lactyl phosphate (LP) group to produce F 420 -0 (Fig. 1A). Finally, mature F 420 coenzymes in different organisms contain 1-7 glutamate residues (F 420 -n) that are covalently linked by amide bonds through their ␣-and/or ␥-carboxylates (1, 15-17). Although F 420 contains a (deaza)riboflavin moiety, its biochemistry is actually more similar to that of NAD(P) ϩ than FMN/FAD. However, F 420 has a lower midpoint potential (Ϫ360 mV) than NAD(P) ϩ (Ϫ320 mV) (13). The biosynthesis of F 420 uses lactate, 4-hydroxyphenylpyruvate (an intermediate in tyrosine biosynthesis), and a pyrimidine intermediate in riboflavin biosynt...

The first step in the biosynthesis of pterins in bacteria and plants is the conversion of GTP to 7,8-dihydro-d-neopterin triphosphate catalyzed by GTP cyclohydrolase I (GTPCHI). Although GTP has been shown to be a precursor of pterins in archaea, homologues of GTPCHI have not been identified in most archaeal genomes. Here we report the identification of a new GTP cyclohydrolase that converts GTP to 7,8-dihydro-d-neopterin 2',3'-cyclic phosphate, the first intermediate in methanopterin biosynthesis in methanogenic archaea. The enzyme from Methanocaldococcus jannaschii is designated MptA to indicate that it catalyzes the first step in the biosynthesis of methanopterin. MptA is the archetype of a new class of GTP cyclohydrolases that catalyzes a series of reactions most similar to that seen with GTPCHI but unique in that the cyclic phosphate is the product. MptA was found to require Fe2+ for activity. Mutation of conserved histidine residues H200N, H293N, and H295N, expected to be involved in Fe2+ binding, resulted in reduced enzymatic activity but no reduction in the amount of bound iron.

F(420) is a flavin-like redox-active coenzyme commonly used by archaea and some eubacteria in a variety of biochemical reactions in methanogenesis, the formation of secondary metabolites, the degradation of nitroaromatic compounds, activation of nitroimidazofurans, and F(420)-dependent photolysis in DNA repair. Coenzyme F(420)-2 biosynthesis from 7,8-didemethyl-8-hydroxy-5-deazariboflavin (Fo) and lactaldehyde involves six enzymatic steps and five proteins (CofA, CofB, CofC, CofD, and CofE). CofE, a F(420)-0:gamma-glutamyl ligase, is responsible for the last two enzymatic steps; it catalyses the GTP-dependent addition of two L-glutamate residues to F(420)-0 to form F(420)-2. CofE is found in archaea, the aerobic actinomycetes, and cyanobacteria. Here, we report the first crystal structure of the apo-F(420)-0:gamma-glutamyl ligase (CofE-AF) from Archaeoglobus fulgidus and its complex with GDP at 2.5 A and 1.35 A resolution, respectively. The structure of CofE-AF reveals a novel protein fold with an intertwined, butterfly-like dimer formed by two-domain monomers. GDP and Mn(2+) are bound within the putative active site in a large groove at the dimer interface. We show that the enzyme adds a glutamate residue to both F(420)-0 and F(420)-1 in two distinct steps. CofE represents the first member of a new structural family of non-ribosomal peptide synthases.