SummaryFungi are a rich source of bioactive secondary metabolites, and mushroom-forming fungi (Agaricomycetes) are especially known for the synthesis of numerous bioactive and often cytotoxic sesquiterpenoid secondary metabolites. Compared with the large number of sesquiterpene synthases identified in plants, less than a handful of unique sesquiterpene synthases have been described from fungi. Here we describe the functional characterization of six sesquiterpene synthases (Cop1 to Cop6) and two terpeneoxidizing cytochrome P450 monooxygenases (Cox1 and Cox2) from Coprinus cinereus. The genes were cloned and, except for cop5, functionally expressed in Escherichia coli and/or Saccharomyces cerevisiae. Cop1 and Cop2 each synthesize germacrene A as the major product. Cop3 was identified as an a-muurolene synthase, an enzyme that has not been described previously, while Cop4 synthesizes d-cadinene as its major product. Cop6 was originally annotated as a trichodiene synthase homologue but instead was found to catalyse the highly specific synthesis of a-cuprenene. Coexpression of cop6 and the two monooxygenase genes next to it yields oxygenated a-cuprenene derivatives, including cuparophenol, suggesting that these genes encode the enzymes for the biosynthesis of antimicrobial quinone sesquiterpenoids (known as lagopodins) that were previously isolated from C. cinereus and other Coprinus species.

Paracoccus denitrificans methylamine dehydrogenase (MADH) is an enzyme containing a quinone cofactor tryptophan tryptophylquinone (TTQ) derived from two tryptophan residues (betaTrp(57) and betaTrp(108)) within the polypeptide chain. During cofactor formation, the two tryptophan residues become covalently linked, and two carbonyl oxygens are added to the indole ring of betaTrp(57). Expression of active MADH from P. denitrificans requires four other genes in addition to those that encode the polypeptides of the MADH alpha(2)beta(2) heterotetramer. One of these, mauG, has been shown to be involved in TTQ biogenesis. It contains two covalently attached c-type hemes but exhibits unusual properties compared to c-type cytochromes and diheme cytochrome c peroxidases, to which it has some sequence similarity. To test the role that MauG may play in TTQ maturation, the predicted proximal histidine to each heme (His(35) and His(205)) has each been mutated to valine, and wild-type MADH was expressed in the background of these two mauG mutants. The resultant MADH has been characterized by mass spectrometry and electrophoretic and kinetic analyses. The majority species is a TTQ biogenesis intermediate containing a monohydroxylated betaTrp(57), suggesting that this is the natural substrate for MauG. Previous work has shown that MADH mutated at the betaTrp(108) position (the non-oxygenated TTQ partner) is predominantly also this intermediate, and work on these mutants is extended and compared to the MADH expressed in the background of the histidine to valine mauG mutations. In this study, it is unequivocally demonstrated that MauG is required to initiate the formation of the TTQ cross-link, the conversion of a single hydroxyl located on betaTrp(57) to a carbonyl, and the incorporation of the second oxygen into the TTQ ring to complete TTQ biogenesis. The properties of MauG, which are atypical of c-type cytochromes, are discussed in the context of these final stages of TTQ biogenesis.

BACKGROUND If liquid-chromatography–multiple-reaction–monitoring mass spectrometry (LC-MRM/MS) could be used in the large-scale preclinical verification of putative biomarkers, it would obviate the need for the development of expensive immunoassays. In addition, the translation of novel biomarkers to clinical use would be accelerated if the assays used in preclinical studies were the same as those used in the clinical laboratory. To validate this approach, we developed a multiplexed assay for the quantification of 2 clinically well-known biomarkers in human plasma, apolipoprotein A-I and apolipoprotein B (apoA-I and apoB). METHODS We used PeptideAtlas to identify candidate peptides. Human samples were denatured with urea or trifluoroethanol, reduced and alkylated, and digested with trypsin. We compared reversed-phase chromatographic separation of peptides with normal flow and microflow, and we normalized endogenous peptide peak areas to internal standard peptides. We evaluated different methods of calibration and compared the final method with a nephelometric immunoassay. RESULTS We developed a final method using trifluoroethanol denaturation, 21-h digestion, normal flow chromatography-electrospray ionization, and calibration with a single normal human plasma sample. For samples injected in duplicate, the method had intraassay CVs <6% and interassay CVs <12% for both proteins, and compared well with immunoassay (n = 47; Deming regression, LC-MRM/MS = 1.17 × immunoassay – 36.6; Sx|y = 10.3 for apoA-I and LC-MRM/MS = 1.21 × immunoassay + 7.0; Sx|y = 7.9 for apoB). CONCLUSIONS Multiplexed quantification of proteins in human plasma/serum by LC-MRM/MS is possible and compares well with clinically useful immunoassays. The potential application of single-point calibration to large clinical studies could simplify efforts to reduce day-to-day digestion variability.

Cyanobacteria are a rich source of natural products and are known to produce terpenoids. These bacteria are the major source of the musty-smelling terpenes geosmin and 2-methylisoborneol, which are found in many natural water supplies; however, no terpene synthases have been characterized from these organisms to date. Here, we describe the characterization of three sesquiterpene synthases identified in Nostoc sp. strain PCC 7120 (terpene synthase NS1) and Nostoc punctiforme PCC 73102 (terpene synthases NP1 and NP2). The second terpene synthase in N. punctiforme (NP2) is homologous to fusion-type sesquiterpene synthases from Streptomyces spp. shown to produce geosmin via an intermediate germacradienol. The enzymes were functionally expressed in Escherichia coli, and their terpene products were structurally identified as germacrene A (from NS1), the eudesmadiene 8a-epi-␣-selinene (from NP1), and germacradienol (from NP2). The product of NP1, 8a-epi-␣-selinene, so far has been isolated only from termites, in which it functions as a defense compound. Terpene synthases NP1 and NS1 are part of an apparent minicluster that includes a P450 and a putative hybrid two-component protein located downstream of the terpene synthases. Coexpression of P450 genes with their adjacent located terpene synthase genes in E. coli demonstrates that the P450 from Nostoc sp. can be functionally expressed in E. coli when coexpressed with a ferredoxin gene and a ferredoxin reductase gene from Nostoc and that the enzyme oxygenates the NS1 terpene product germacrene A. This represents to the best of our knowledge the first example of functional expression of a cyanobacterial P450 in E. coli.Terpenoids constitute the largest group of natural products, constituting over 20,000 described compounds (14). These compounds have a wide range of biological functions and are synthesized by plants and microbes as, for example, pigments, hormones and signaling molecules, antibiotics, antifeedants, or pollinator attractants. Many of these compounds are present in minute quantities and have been an important target for synthetic chemists (22, 52) and metabolic engineers (10,35,50).Terpenes are synthesized from linear isoprene diphosphate precursors with various chain lengths, and their enormous structural diversity is the result of the large number of possible enzyme-catalyzed cyclizations and rearrangements that the double-bond-containing isoprene chains can undergo (14, 49). Cyclizations of isoprene diphosphate chains are catalyzed by terpene synthases (also known as terpene cyclases). Depending on the chain length of their isoprene diphosphate substrates, terpene synthases can be classified as mono-, sesqui-, or diterpene synthases, which catalyze the cyclization of geranyl diphosphate (C 10 ), farnesyl diphosphate (FPP; C 15 ), or geranylgeranyl diphosphate, respectively. Well-known examples of cyclic terpenes are the monoterpene 2-methylisoborneol and sesquiterpene geosmin (responsible for the earthy odor of soil and lake water), the trichothecenes (m...