The Function of Two Radical-SAM Enzymes, HcgA and HcgG, in the Biosynthesis of the [Fe]-Hydrogenase Cofactor.

In the biosynthesis of the iron-guanylylpyridinol (FeGP) cofactor, 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol (1) is 3-methylated to form 2, then 4-guanylylated to form 3, and converted into the full cofactor. HcgA-G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S-adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis ΔhcgA strain was complemented with HcgA or precursors 1, 2 or 3. The results suggested that HcgA catalyzes the biosynthetic reaction that forms 1. We demonstrated the formation of 1 by HcgA using the 3 kDa cell extract filtrate as the substrate. Biosynthesis in the ΔhcgG system was recovered by HcgG but not by 3, which indicated that HcgG catalyzes the reactions after the biosynthesis of 3. The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor.

Two Different Quinohemoprotein Amine Dehydrogenases Initiate Anaerobic Degradation of Aromatic Amines in Aromatoleum aromaticum EbN1.

Aromatic amines like 2-phenylethylamine (2-PEA) and benzylamine (BAm) have been identified as novel growth substrates of the betaproteobacterium Aromatoleum aromaticum EbN1, which degrades a wide variety of aromatic compounds in the absence of oxygen under denitrifying growth conditions. The catabolic pathway of these amines was identified, starting with their oxidative deamination to the corresponding aldehydes, which are then further degraded via the enzymes of the phenylalanine or benzyl alcohol metabolic pathways. Two different periplasmic quinohemoprotein amine dehydrogenases involved in 2-PEA or BAm metabolism were identified and characterized. Both enzymes consist of three subunits, contain two heme c cofactors in their α-subunits, and exhibit extensive processing of their γ-subunits, generating four intramolecular thioether bonds and a cysteine tryptophylquinone (CTQ) cofactor. One of the enzymes was present in cells grown with 2-PEA or other substrates, showed an α2ß2γ2 composition, and had a rather broad substrate spectrum, which included 2-PEA, BAm, tyramine, and 1-butylamine. In contrast, the other enzyme was specifically induced in BAm-grown cells, showing an αßγ composition and activity only with BAm and 2-PEA. Since the former enzyme showed the highest catalytic efficiency with 2-PEA and the latter with BAm, they were designated 2-PEADH and benzylamine dehydrogenase (BAmDH). The catalytic properties and inhibition patterns of 2-PEADH and BAmDH showed considerable differences and were compared to previously characterized quinohemoproteins of the same enzyme family.IMPORTANCE The known substrate spectrum of A. aromaticum EbN1 is expanded toward aromatic amines, which are metabolized as sole substrates coupled to denitrification. The characterization of the two quinohemoprotein isoenzymes involved in degrading either 2-PEA or BAm expands the knowledge of this enzyme family and establishes for the first time that the necessary maturation of their quinoid CTQ cofactors does not require the presence of molecular oxygen. Moreover, the study revealed a highly interesting regulatory phenomenon, suggesting that growth with BAm leads to a complete replacement of 2-PEADH by BAmDH, which has considerably different catalytic and inhibition properties.

Mating-Induced Differential Peptidomics of Neuropeptides and Protein Hormones in Agrotis ipsilon Moths.

In many insects, mating induces drastic changes in male and female responses to sex pheromones or host-plant odors. In the male moth Agrotis ipsilon, mating induces a transient inhibition of behavioral and neuronal responses to the female sex pheromone. As neuropeptides and peptide hormones regulate most behavioral processes, we hypothesize that they could be involved in this mating-dependent olfactory plasticity. Here we used next-generation RNA sequencing and a combination of liquid chromatography, matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and direct tissue profiling to analyze the transcriptome and peptidome of different brain compartments in virgin and mated males and females of A. ipsilon. We identified 37 transcripts encoding putative neuropeptide precursors and 54 putative bioactive neuropeptides from 23 neuropeptide precursors (70 sequences in total, 25 neuropeptide precursors) in different areas of the central nervous system including the antennal lobes, the gnathal ganglion, and the corpora cardiaca-corpora allata complex. Comparisons between virgin and mated males and females revealed tissue-specific differences in peptide composition between sexes and according to physiological state. Mated males showed postmating differences in neuropeptide occurrence, which could participate in the mating-induced olfactory plasticity.

Phylogenetic and Structural Comparisons of the Three Types of Methyl Coenzyme M Reductase from Methanococcales and Methanobacteriales.

The phylogenetically diverse family of methanogenic archaea universally use methyl coenzyme M reductase (MCR) for catalyzing the final methane-forming reaction step of the methanogenic energy metabolism. Some methanogens of the orders Methanobacteriales and Methanococcales contain two isoenzymes. Comprehensive phylogenetic analyses on the basis of all three subunits grouped MCRs from Methanobacteriales and Methanococcales into three distinct types: (i) MCRs from Methanobacteriales, (ii) MCRs from Methanobacteriales and Methanococcales, and (iii) MCRs from Methanococcales The first and second types contain MCR isoenzymes I and II from Methanothermobacter marburgensis, respectively; therefore, they were designated MCR type I and type II and accordingly; the third one was designated MCR type III. For comparison with the known MCR type I and type II structures, we determined the structure of MCR type III from Methanotorris formicicus and Methanothermococcus thermolithotrophicus As predicted, the three MCR types revealed highly similar overall structures and virtually identical active site architectures reflecting the chemically challenging mechanism of methane formation. Pronounced differences were found at the protein surface with respect to loop geometries and electrostatic properties, which also involve the entrance of the active-site funnel. In addition, the C-terminal end of the γ-subunit is prolonged by an extra helix after helix γ8 in MCR type II and type III, which is, however, differently arranged in the two MCR types. MCR types I, II, and III share most of the posttranslational modifications which appear to fine-tune the enzymatic catalysis. Interestingly, MCR type III lacks the methyl-cysteine but possesses in subunit α of M. formicicus a 6-hydroxy-tryptophan, which thus far has been found only in the α-amanitin toxin peptide but not in proteins.IMPORTANCE Methyl coenzyme M reductase (MCR) represents a prime target for the mitigation of methane releases. Phylogenetic analyses of MCRs suggested several distinct sequence clusters; those from Methanobacteriales and Methanococcales were subdivided into three types: MCR type I from Methanobacteriales, MCR type II from Methanobacteriales and Methanococcales, and the newly designated MCR type III exclusively from Methanococcales We determined the first X-ray structures for an MCR type III. Detailed analyses revealed substantial differences between the three types only in the peripheral region. The subtle modifications identified and electrostatic profiles suggested enhanced substrate binding for MCR type III. In addition, MCR type III from Methanotorris formicicus contains 6-hydroxy-tryptophan, a new posttranslational modification that thus far has been found only in the α-amanitin toxin.

Identification of HcgC as a SAM-Dependent Pyridinol Methyltransferase in [Fe]-Hydrogenase Cofactor Biosynthesis.

Previous retrosynthetic and isotope-labeling studies have indicated that biosynthesis of the iron guanylylpyridinol (FeGP) cofactor of [Fe]-hydrogenase requires a methyltransferase. This hypothetical enzyme covalently attaches the methyl group at the 3-position of the pyridinol ring. We describe the identification of HcgC, a gene product of the hcgA-G cluster responsible for FeGP cofactor biosynthesis. It acts as an S-adenosylmethionine (SAM)-dependent methyltransferase, based on the crystal structures of HcgC and the HcgC/SAM and HcgC/S-adenosylhomocysteine (SAH) complexes. The pyridinol substrate, 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol, was predicted based on properties of the conserved binding pocket and substrate docking simulations. For verification, the assumed substrate was synthesized and used in a kinetic assay. Mass spectrometry and NMR analysis revealed 6-carboxymethyl-3,5-dimethyl-4-hydroxy-2-pyridinol as the reaction product, which confirmed the function of HcgC.

Protein-pyridinol thioester precursor for biosynthesis of the organometallic acyl-iron ligand in [Fe]-hydrogenase cofactor.

The iron-guanylylpyridinol (FeGP) cofactor of [Fe]-hydrogenase contains a prominent iron centre with an acyl-Fe bond and is the only acyl-organometallic iron compound found in nature. Here, we identify the functions of HcgE and HcgF, involved in the biosynthesis of the FeGP cofactor using structure-to-function strategy. Analysis of the HcgE and HcgF crystal structures with and without bound substrates suggest that HcgE catalyses the adenylylation of the carboxy group of guanylylpyridinol (GP) to afford AMP-GP, and subsequently HcgF catalyses the transesterification of AMP-GP to afford a Cys (HcgF)-S-GP thioester. Both enzymatic reactions are confirmed by in vitro assays. The structural data also offer plausible catalytic mechanisms. This strategy of thioester activation corresponds to that used for ubiquitin activation, a key event in the regulation of multiple cellular processes. It further implicates a nucleophilic attack onto the acyl carbon presumably via an electron-rich Fe(0)- or Fe(I)-carbonyl complex in the Fe-acyl formation.

A secreted Ustilago maydis effector promotes virulence by targeting anthocyanin biosynthesis in maize.

The biotrophic fungus Ustilago maydis causes smut disease in maize with characteristic tumor formation and anthocyanin induction. Here, we show that anthocyanin biosynthesis is induced by the virulence promoting secreted effector protein Tin2. Tin2 protein functions inside plant cells where it interacts with maize protein kinase ZmTTK1. Tin2 masks a ubiquitin-proteasome degradation motif in ZmTTK1, thus stabilizing the active kinase. Active ZmTTK1 controls activation of genes in the anthocyanin biosynthesis pathway. Without Tin2, enhanced lignin biosynthesis is observed in infected tissue and vascular bundles show strong lignification. This is presumably limiting access of fungal hyphae to nutrients needed for massive proliferation. Consistent with this assertion, we observe that maize brown midrib mutants affected in lignin biosynthesis are hypersensitive to U. maydis infection. We speculate that Tin2 rewires metabolites into the anthocyanin pathway to lower their availability for other defense responses. DOI: http://dx.doi.org/10.7554/eLife.01355.001.

FlaX, a unique component of the crenarchaeal archaellum, forms oligomeric ring-shaped structures and interacts with the motor ATPase FlaI.

Archaella are the archaeal motility structure, which are structurally similar to gram-negative bacterial type IV pili but functionally resemble bacterial flagella. Structural and biochemical data of archaellum subunits are missing. FlaX, a conserved subunit in crenarchaeal archaella, formed high molecular weight complexes that adapted a ring-like structure with an approximate diameter of 30 nm. The C terminus of FlaX was not only involved in the oligomerization, but also essential for FlaX interaction with FlaI, the bifunctional ATPase that is involved in assembly and rotation of the archaellum. This study gives first insights in the assembly apparatus of archaella.

Peptidomics of the agriculturally damaging larval stage of the cabbage root fly Delia radicum (Diptera: Anthomyiidae).

The larvae of the cabbage root fly induce serious damage to cultivated crops of the family Brassicaceae. We here report the biochemical characterisation of neuropeptides from the central nervous system and neurohemal organs, as well as regulatory peptides from enteroendocrine midgut cells of the cabbage maggot. By LC-MALDI-TOF/TOF and chemical labelling with 4-sulfophenyl isothiocyanate, 38 peptides could be identified, representing major insect peptide families: allatostatin A, allatostatin C, FMRFamide-like peptides, kinin, CAPA peptides, pyrokinins, sNPF, myosuppressin, corazonin, SIFamide, sulfakinins, tachykinins, NPLP1-peptides, adipokinetic hormone and CCHamide 1. We also report a new peptide (Yamide) which appears to be homolog to an amidated eclosion hormone-associated peptide in several Drosophila species. Immunocytochemical characterisation of the distribution of several classes of peptide-immunoreactive neurons and enteroendocrine cells shows a very similar but not identical peptide distribution to Drosophila. Since peptides regulate many vital physiological and behavioural processes such as moulting or feeding, our data may initiate the pharmacological testing and development of new specific peptide-based protection methods against the cabbage root fly and its larva.

Biosynthesis of the iron-guanylylpyridinol cofactor of [Fe]-hydrogenase in methanogenic archaea as elucidated by stable-isotope labeling.

[Fe]-hydrogenase catalyzes the reversible hydride transfer from H(2) to methenyltetrahydromethanoptherin, which is an intermediate in methane formation from H(2) and CO(2) in methanogenic archaea. The enzyme harbors a unique active site iron-guanylylpyridinol (FeGP) cofactor, in which a low-spin Fe(II) is coordinated by a pyridinol-N, an acyl group, two carbon monoxide, and the sulfur of the enzyme's cysteine. Here, we studied the biosynthesis of the FeGP cofactor by following the incorporation of (13)C and (2)H from labeled precursors into the cofactor in growing methanogenic archaea and by subsequent NMR, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) and IR analysis of the isolated cofactor and reference compounds. The pyridinol moiety of the cofactor was found to be synthesized from three C-1 of acetate, two C-2 of acetate, two C-1 of pyruvate, one carbon from the methyl group of l-methionine, and one carbon directly from CO(2). The metabolic origin of the two CO-ligands was CO(2) rather than C-1 or C-2 of acetate or pyruvate excluding that the two CO are derived from dehydroglycine as has previously been shown for the CO-ligands in [FeFe]-hydrogenases. A formation of CO from CO(2) via direct reduction catalyzed by a nickel-dependent CO dehydrogenase or from formate could also be excluded. When the cells were grown in the presence of (13)CO, the two CO-ligands and the acyl group became (13)C-labeled, indicating either that free CO is an intermediate in their synthesis or that free CO can exchange with these iron-bound ligands. Based on these findings, we propose pathways for how the FeGP cofactor might be synthesized.

Evidence for acyl-iron ligation in the active site of [Fe]-hydrogenase provided by mass spectrometry and infrared spectroscopy.

[Fe]-hydrogenase catalyzes the reversible heterolytic cleavage of H(2) and stereo-specific hydride transfer to the substrate methenyltetrahydromethanopterin in methanogenic archaea. This enzyme contains a unique iron guanylylpyridinol (FeGP) cofactor as a prosthetic group. It has recently been proposed-on the basis of crystal structural analyses of the [Fe]-hydrogenase holoenzyme-that the FeGP cofactor contains an acyl-iron ligation, the first one reported in a biological system. We report here that the cofactor can be reversibly extracted with acids; its exact mass has been determined by electrospray ionization Fourier transform ion cyclotron resonance mass-spectrometry. The measured mass of the intact cofactor and its gas-phase fragments are consistent with the proposed structure. The mass of the light decomposition products of the cofactor support the presence of acyl-iron ligation. Attenuated total reflection infrared spectroscopy of the FeGP cofactor revealed a band near wave number 1700 cm(-1), which was assigned to the C=O (double bond) stretching mode of the acyl-iron ligand.

Structure of a methyl-coenzyme M reductase from Black Sea mats that oxidize methane anaerobically.

The anaerobic oxidation of methane (AOM) with sulphate, an area currently generating great interest in microbiology, is accomplished by consortia of methanotrophic archaea (ANME) and sulphate-reducing bacteria. The enzyme activating methane in methanotrophic archaea has tentatively been identified as a homologue of methyl-coenzyme M reductase (MCR) that catalyses the methane-forming step in methanogenic archaea. Here we report an X-ray structure of the 280 kDa heterohexameric ANME-1 MCR complex. It was crystallized uniquely from a protein ensemble purified from consortia of microorganisms collected with a submersible from a Black Sea mat catalysing AOM with sulphate. Crystals grown from the heterogeneous sample diffract to 2.1 Å resolution and consist of a single ANME-1 MCR population, demonstrating the strong selective power of crystallization. The structure revealed ANME-1 MCR in complex with coenzyme M and coenzyme B, indicating the same substrates for MCR from methanotrophic and methanogenic archaea. Differences between the highly similar structures of ANME-1 MCR and methanogenic MCR include a F(430) modification, a cysteine-rich patch and an altered post-translational amino acid modification pattern, which may tune the enzymes for their functions in different biological contexts.

Metabolic priming by a secreted fungal effector.

Maize smut caused by the fungus Ustilago maydis is a widespread disease characterized by the development of large plant tumours. U. maydis is a biotrophic pathogen that requires living plant tissue for its development and establishes an intimate interaction zone between fungal hyphae and the plant plasma membrane. U. maydis actively suppresses plant defence responses by secreted protein effectors. Its effector repertoire comprises at least 386 genes mostly encoding proteins of unknown function and expressed exclusively during the biotrophic stage. The U. maydis secretome also contains about 150 proteins with probable roles in fungal nutrition, fungal cell wall modification and host penetration as well as proteins unlikely to act in the fungal-host interface like a chorismate mutase. Chorismate mutases are key enzymes of the shikimate pathway and catalyse the conversion of chorismate to prephenate, the precursor for tyrosine and phenylalanine synthesis. Root-knot nematodes inject a secreted chorismate mutase into plant cells likely to affect development. Here we show that the chorismate mutase Cmu1 secreted by U. maydis is a virulence factor. The enzyme is taken up by plant cells, can spread to neighbouring cells and changes the metabolic status of these cells through metabolic priming. Secreted chorismate mutases are found in many plant-associated microbes and might serve as general tools for host manipulation.

A seven-WD40 protein related to human RACK1 regulates mating and virulence in Ustilago maydis.

In mammalian cells RACK1 serves as a scaffold protein that has a role in integrating inputs from different signalling pathways and affects translation through association with ribosomes. Ustilago maydis contains a seven-WD40 repeat motif protein designated Rak1, which shows 68% identity to RACK1 and 51% identity to Asc1p of Saccharomyces cerevisiae. An asc1 mutant could be complemented by introduction of U. maydis rak1. The deletion of rak1 affected cell growth, cell wall integrity and specifically attenuated cell fusion. This latter defect was caused by reduced expression of prf1 encoding the regulator for pheromone (mfa) and pheromone-receptor genes. Rak1 interacts with a variety of ribosomal proteins and microarray analysis revealed that the deletion of rak1 led to severely reduced expression of rop1, a transcriptional activator of prf1. The constitutive expression of rop1 could rescue the defect of mfa1 expression as well as conjugation tube formation in response to pheromone induction in the rak1 mutant. Moreover, a solopathogenic rak1 mutant failed to respond to plant-derived stimuli, resulting in attenuated filamentation and pathogenicity. This could be partially rescued by constitutive expression of the b heterodimer. These data suggest that rak1 is a regulator of rop1 expression with additional roles after cell fusion.

Peptidomics and peptide hormone processing in the Drosophila midgut.

Peptide hormones are key messengers in the signaling network between the nervous system, endocrine glands, energy stores and the gastrointestinal tract that regulates feeding and metabolism. Studies on the Drosophila nervous system have uncovered parallels and homologies in homeostatic peptidergic signaling between fruit flies and vertebrates. Yet, the role of enteroendocrine peptides in the regulation of feeding and metabolism has not been explored, with research hampered by the unknown identity of peptides produced by the fly's intestinal tract. We performed a peptidomic LC/MS analysis of the fruit fly midgut containing the enteroendocrine cells. By MS/MS fragmentation, we found 24 peptides from 9 different preprohormones in midgut extracts, including MIP-4 and 2 forms of AST-C. DH(31), CCHamide1 and CCHamide2 are biochemically characterized for the first time. All enteroendocrine peptides represent brain-gut peptides, and apparently are processed by Drosophila prohormone convertase 2 (AMON) as suggested by impaired peptide detectability in amon mutants and localization of amon-driven GFP to enteroendocrine cells. Because of its genetic amenability and peptide diversity, Drosophila provides a good model system to study peptide signaling. The identification of enteroendocrine peptides in the fruit fly provides a platform to address functions of gut peptide hormones in the regulation of feeding and metabolism.

Post-translational modifications in the active site region of methyl-coenzyme M reductase from methanogenic and methanotrophic archaea.

Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaea. Isoenzyme I from Methanothermobacter marburgensiswas shown to contain a thioxo peptide bond and four methylated amino acids in the active site region. We report here that MCRs from all methanogens investigated contain the thioxo peptide bond, but that the enzymes differ in their post-translational methylations. The MS analysis included MCR I and MCR II from Methanothermobacter marburgensis, MCR I from Methanocaldococcus jannaschii and Methanoculleus thermophilus, and MCR from Methanococcus voltae, Methanopyrus kandleri and Methanosarcina barkeri. Two MCRs isolated from Black Sea mats containing mainly methanotrophic archaea of the ANME-1 cluster were also analyzed.

Direct peptide profiling of lateral cell groups of the antennal lobes of Manduca sexta reveals specific composition and changes in neuropeptide expression during development.

The paired antennal lobes are the first integration centers for odor information in the insect brain. In the sphinx moth Manduca sexta, like in other holometabolous insects, they are formed during metamorphosis. To further understand mechanisms involved in the formation of this particularly well investigated brain area, we performed a direct peptide profiling of a well defined cell group (the lateral cell group) of the antennal lobe throughout development by MALDI-TOF mass spectrometry. Although the majority of the about 100 obtained ion signals represent still unknown substances, this first peptidomic characterization of this cell group indicated the occurrence of 12 structurally known neuropeptides. Among these peptides are helicostatin 1, cydiastatins 2, 3, and 4, M. sexta-allatotropin (Mas-AT), M. sexta-FLRFamide (Mas-FLRFamide) I, II, and III, nonblocked Mas-FLRFamide I, and M. sexta-myoinhibitory peptides (Mas-MIPs) III, V, and VI. The identity of two of the allatostatins (cydiastatins 3 and 4) and Mas-AT were confirmed by tandem mass spectrometry (MALDI-TOF/TOF). During development of the antennal lobe, number and frequency of ion signals including those representing known peptides generally increased at the onset of glomeruli formation at pupal Stage P7/8, with cydiastatin 2, helicostatin 1, and Mas-MIP V being the exceptions. Cydiastatin 2 showed transient occurrence mainly during the period of glomerulus formation, helicostatin 1 was restricted to late pupae and adults, while Mas-MIP V occurred exclusively in adult antennal lobes. The power of the applied direct mass spectrometric profiling lies in the possibility of chemically identifying neuropeptides of a given cell population in a fast and reliable manner, at any developmental stage in single specimens. The identification of neuropeptides in the antennal lobes now allows to specifically address the function of these signaling molecules during the formation of the antennal lobe network.

A simple purification protocol for the detection of peptide hormones in the hemolymph of individual insects by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The endocrine system of insects is largely based on peptide hormones. Nevertheless, an unequivocal chemical demonstration of the occurence in the hemolymph (the 'insect blood') is still lacking for most if not all insect peptide hormones, although this is the only way to prove their hormonal status. Focusing on peptides released during ecdysis behavior of the tobacco hornworm Manduca sexta, we developed a purification protocol based on ultrafiltration and a single reversed-phase high-performance liquid chromatography (RP-HPLC) step that for the first time allowed the mass spectrometric and chemical identification of a peptide hormone in the hemolymph of single specimens. Since this method is simple, relatively cheap and fast, it should be useful for routine endocrinological analyses and for monitoring peptide release during different physiological conditions and behaviors in insects.

Heme biosynthesis in Methanosarcina barkeri via a pathway involving two methylation reactions.

The methanogenic archaeon Methanosarcina barkeri synthesizes protoheme via precorrin-2, which is formed from uroporphyrinogen III in two consecutive methylation reactions utilizing S-adenosyl-L-methionine. The existence of this pathway, previously exclusively found in the sulfate-reducing delta-proteobacterium Desulfovibrio vulgaris, was demonstrated for M. barkeri via the incorporation of two methyl groups from methionine into protoheme.