Structural Dynamics of the Methyl-Coenzyme M Reductase Active Site Are Influenced by Coenzyme F430 Modifications.

Methyl-coenzyme M reductase (MCR) is a central player in methane biogeochemistry, governing methanogenesis and the anaerobic oxidation of methane (AOM) in methanogens and anaerobic methanotrophs (ANME), respectively. The prosthetic group of MCR is coenzyme F430, a nickel-containing tetrahydrocorphin. Several modified versions of F430 have been discovered, including the 172-methylthio-F430 (mtF430) used by ANME-1 MCR. Here, we employ molecular dynamics (MD) simulations to investigate the active site dynamics of MCR from Methanosarcina acetivorans and ANME-1 when bound to the canonical F430 compared to 172-thioether coenzyme F430 variants and substrates (methyl-coenzyme M and coenzyme B) for methane formation. Our simulations highlight the importance of the Gln to Val substitution in accommodating the 172 methylthio modification in ANME-1 MCR. Modifications at the 172 position disrupt the canonical substrate positioning in M. acetivorans MCR. However, in some replicates, active site reorganization to maintain substrate positioning suggests that the modified F430 variants could be accommodated in a methanogenic MCR. We additionally report the first quantitative estimate of MCR intrinsic electric fields that are pivotal in driving methane formation. Our results suggest that the electric field aligned along the CH3-S-CoM thioether bond facilitates homolytic bond cleavage, coinciding with the proposed catalytic mechanism. Structural perturbations, however, weaken and misalign these electric fields, emphasizing the importance of the active site structure in maintaining their integrity. In conclusion, our results deepen the understanding of MCR active site dynamics, the enzyme's organizational role in intrinsic electric fields for catalysis, and the interplay between active site structure and electrostatics.

Control of biofilm formation by an Agrobacterium tumefaciens pterin-binding periplasmic protein conserved among diverse Proteobacteria.

Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen Agrobacterium tumefaciens produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase, is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are produced via a nonessential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering c-di-GMP breakdown and dampening its synthesis. Pterins are excreted, and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a pruA mutant. PruR and DcpA are encoded in an operon with wide conservation among diverse Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a pterin-responsive regulatory mechanism that controls biofilm formation and related c-di-GMP-dependent phenotypes in A. tumefaciens and potentially acts more widely in multiple proteobacterial lineages.

Biochemical and genetic studies define the functions of methylthiotransferases in methanogenic and methanotrophic archaea.

Methylthiotransferases (MTTases) are radical S-adenosylmethionine (SAM) enzymes that catalyze the addition of a methylthio (-SCH3) group to an unreactive carbon center. These enzymes are responsible for the production of 2-methylthioadenosine (ms2A) derivatives found at position A37 of select tRNAs in all domains of life. Additionally, some bacteria contain the RimO MTTase that catalyzes the methylthiolation of the S12 ribosomal protein. Although the functions of MTTases in bacteria and eukaryotes have been established via detailed genetic and biochemical studies, MTTases from the archaeal domain of life are understudied and the substrate specificity determinants of MTTases remain unclear. Here, we report the in vitro enzymatic activities of an MTTase (C4B56_06395) from a thermophilic Ca. Methanophagales anaerobic methanotroph (ANME) as well as the MTTase from a hyperthermophilic methanogen - MJ0867 from Methanocaldococcus jannaschii. Both enzymes catalyze the methylthiolation of N6-threonylcarbamoyladenosine (t6A) and N6-hydroxynorvalylcarbamoyladenosine (hn6A) residues to produce 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A) and 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine (ms2hn6A), respectively. To further assess the function of archaeal MTTases, we analyzed select tRNA modifications in a model methanogen - Methanosarcina acetivorans - and generated a deletion of the MTTase-encoding gene (MA1153). We found that M. acetivorans produces ms2hn6A in exponential phase of growth, but does not produce ms2t6A in detectable amounts. Upon deletion of MA1153, the ms2A modification was absent, thus confirming the function of MtaB-family MTTases in generating ms2hn6A modified nucleosides in select tRNAs.

Control of Biofilm Formation by an Agrobacterium tumefaciens Pterin-Binding Periplasmic Protein Conserved Among Pathogenic Bacteria.

Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen Agrobacterium tumefaciens produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (cdGMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase (DGC-PDE), is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are formed via a non-essential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering cdGMP breakdown and dampening its synthesis. Pterins are excreted and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a pruA mutant. PruR and DcpA are encoded in an operon that is conserved amongst multiple Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a new pterin-responsive regulatory mechanism that controls biofilm formation and related cdGMP-dependent phenotypes in A. tumefaciens and is found in multiple additional bacterial pathogens.

A framework of transient hypercapnia to achieve an increased cerebral blood flow induced by nasal breathing during aerobic exercise.

During exercise, cerebral blood flow (CBF) is expected to only increase to a maximal volume up to a moderate intensity aerobic effort, suggesting that CBF is expected to decline past 70 % of a maximal aerobic effort. Increasing CBF during exercise permits an increased cerebral metabolic activity that stimulates neuroplasticity and other key processes of cerebral adaptations that ultimately improve cognitive health. Recent work has focused on utilizing gas-induced exposure to intermittent hypoxia during aerobic exercise to maximize the improvements in cognitive function compared to those seen under normoxic conditions. However, it is postulated that exercising by isolating breathing only to the nasal route may provide a similar effect by stimulating a transient hypercapnic condition that is non-gas dependent. Because nasal breathing prevents hyperventilation during exercise, it promotes an increase in the partial arterial pressure of CO2. The rise in systemic CO2 stimulates hypercapnia and permits the upregulation of hypoxia-related genes. In addition, the rise in systemic CO2 stimulates cerebral vasodilation, promoting a greater increase in CBF than seen during normoxic conditions. While more research is warranted, nasal breathing might also promote benefits related to improved sleep, greater immunity, and body fat loss. Altogether, this narrative review presents a theoretical framework by which exercise-induced hypercapnia by utilizing nasal breathing during moderate-intensity aerobic exercise may promote greater health adaptations and cognitive improvements than utilizing oronasal breathing.

The Chlamydia trachomatis p-aminobenzoate synthase CADD is a manganese-dependent oxygenase that uses its own amino acid residues as substrates.

CADD (chlamydia protein associating with death domains) is a p-aminobenzoate (pAB) synthase involved in a noncanonical route for tetrahydrofolate biosynthesis in Chlamydia trachomatis. Although previously implicated to employ a diiron cofactor, here, we show that pAB synthesis by CADD requires manganese and the physiological cofactor is most likely a heterodinuclear Mn/Fe cluster. Isotope-labeling experiments revealed that the two oxygen atoms in the carboxylic acid portion of pAB are derived from molecular oxygen. Further, mass spectrometry-based proteomic analyses of CADD-derived peptides demonstrated a glycine substitution at Tyr27, providing strong evidence that this residue is sacrificed for pAB synthesis. Additionally, Lys152 was deaminated and oxidized to aminoadipic acid, supporting its proposed role as a sacrificial amino group donor.

Highlighting the Unique Roles of Radical S-Adenosylmethionine Enzymes in Methanogenic Archaea.

Radical S-adenosylmethionine (SAM) enzymes catalyze an impressive variety of difficult biochemical reactions in various pathways across all domains of life. These metalloenzymes employ a reduced [4Fe-4S] cluster and SAM to generate a highly reactive 5'-deoxyadenosyl radical that is capable of initiating catalysis on otherwise unreactive substrates. Interestingly, the genomes of methanogenic archaea encode many unique radical SAM enzymes with underexplored or completely unknown functions. These organisms are responsible for the yearly production of nearly 1 billion tons of methane, a potent greenhouse gas as well as a valuable energy source. Thus, understanding the details of methanogenic metabolism and elucidating the functions of essential enzymes in these organisms can provide insights into strategies to decrease greenhouse gas emissions as well as inform advances in bioenergy production processes. This minireview provides an overview of the current state of the field regarding the functions of radical SAM enzymes in methanogens and discusses gaps in knowledge that should be addressed.

Overview of Diverse Methyl/Alkyl-Coenzyme M Reductases and Considerations for Their Potential Heterologous Expression.

Methyl-coenzyme M reductase (MCR) is an archaeal enzyme that catalyzes the final step of methanogenesis and the first step in the anaerobic oxidation of methane, the energy metabolisms of methanogens and anaerobic methanotrophs (ANME), respectively. Variants of MCR, known as alkyl-coenzyme M reductases, are involved in the anaerobic oxidation of short-chain alkanes including ethane, propane, and butane as well as the catabolism of long-chain alkanes from oil reservoirs. MCR is a dimer of heterotrimers (encoded by mcrABG) and requires the nickel-containing tetrapyrrole prosthetic group known as coenzyme F430. MCR houses a series of unusual post-translational modifications within its active site whose identities vary depending on the organism and whose functions remain unclear. Methanogenic MCRs are encoded in a highly conserved mcrBDCGA gene cluster, which encodes two accessory proteins, McrD and McrC, that are believed to be involved in the assembly and activation of MCR, respectively. The requirement of a unique and complex coenzyme, various unusual post-translational modifications, and many remaining questions surrounding assembly and activation of MCR largely limit in vitro experiments to native enzymes with recombinant methods only recently appearing. Production of MCRs in a heterologous host is an important step toward developing optimized biocatalytic systems for methane production as well as for bioconversion of methane and other alkanes into value-added compounds. This review will first summarize MCR catalysis and structure, followed by a discussion of advances and challenges related to the production of diverse MCRs in a heterologous host.

Lysine 2,3-Aminomutase and a Newly Discovered Glutamate 2,3-Aminomutase Produce ß-Amino Acids Involved in Salt Tolerance in Methanogenic Archaea.

Many methanogenic archaea synthesize ß-amino acids as osmolytes that allow survival in high salinity environments. Here, we investigated the radical S-adenosylmethionine (SAM) aminomutases involved in the biosynthesis of Nε-acetyl-ß-lysine and ß-glutamate in Methanococcus maripaludis C7. Lysine 2,3-aminomutase (KAM), encoded by MmarC7_0106, was overexpressed and purified from Escherichia coli, followed by biochemical characterization. In the presence of l-lysine, SAM, and dithionite, this archaeal KAM had a kcat = 14.3 s-1 and a Km = 19.2 mM. The product was shown to be 3(S)-ß-lysine, which is like the well-characterized Clostridium KAM as opposed to the E. coli KAM that produces 3(R)-ß-lysine. We further describe the function of MmarC7_1783, a putative radical SAM aminomutase with a ∼160 amino acid extension at its N-terminus. Bioinformatic analysis of the possible substrate-binding residues suggested a function as glutamate 2,3-aminomutase, which was confirmed here through heterologous expression in a methanogen followed by detection of ß-glutamate in cell extracts. ß-Glutamate has been known to serve as an osmolyte in select methanogens for a long time, but its biosynthetic origin remained unknown until now. Thus, this study defines the biosynthetic routes for ß-lysine and ß-glutamate in M. maripaludis and expands the importance and diversity of radical SAM enzymes in all domains of life.

Biogenic formation of amorphous carbon by anaerobic methanotrophs and select methanogens.

Elemental carbon exists in different structural forms including graphite, diamond, fullerenes, and amorphous carbon. In nature, these materials are produced through abiotic chemical processes under high temperature and pressure but are considered generally inaccessible to biochemical synthesis or breakdown. Here, we identified and characterized elemental carbon isolated from consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB), which together carry out the anaerobic oxidation of methane (AOM). Two different AOM consortia, ANME-1a/HotSeep-1 and ANME-2a/c/Seep-SRB, produce a black material with similar characteristics to disordered graphite and amorphous carbon. Stable isotope probing studies revealed that the carbon is microbially generated during AOM. In addition, we found that select methanogens also produce amorphous carbon with similar characteristics to the carbon from AOM consortia. Biogenic amorphous carbon may serve as a conductive element to facilitate electron transfer, or redox active functional groups associated with the carbon could act as electron donors and acceptors.

An Unusual Route for p-Aminobenzoate Biosynthesis in Chlamydia trachomatis Involves a Probable Self-Sacrificing Diiron Oxygenase.

Chlamydia trachomatis lacks the canonical genes required for the biosynthesis of p-aminobenzoate (pABA), a component of essential folate cofactors. Previous studies revealed a single gene from C. trachomatis, the CT610 gene, that rescues Escherichia coli ΔpabA, ΔpabB, and ΔpabC mutants, which are otherwise auxotrophic for pABA. CT610 shares low sequence similarity to nonheme diiron oxygenases, and the previously solved crystal structure revealed a diiron active site. Genetic studies ruled out several potential substrates for CT610-dependent pABA biosynthesis, including chorismate and other shikimate pathway intermediates, leaving the actual precursor(s) unknown. Here, we supplied isotopically labeled potential precursors to E. coli ΔpabA cells expressing CT610 and found that the aromatic portion of tyrosine was highly incorporated into pABA, indicating that tyrosine is a precursor for CT610-dependent pABA biosynthesis. Additionally, in vitro enzymatic experiments revealed that purified CT610 exhibits low pABA synthesis activity under aerobic conditions in the absence of tyrosine or other potential substrates, where only the addition of a reducing agent such as dithiothreitol appears to stimulate pABA production. Furthermore, site-directed mutagenesis studies revealed that two conserved active site tyrosine residues are essential for the pABA synthesis reaction in vitro Thus, the current data are most consistent with CT610 being a unique self-sacrificing enzyme that utilizes its own active site tyrosine residue(s) for pABA biosynthesis in a reaction that requires O2 and a reduced diiron cofactor.IMPORTANCEChlamydia trachomatis is the most reported sexually transmitted infection in the United States and the leading cause of infectious blindness worldwide. Unlike many other intracellular pathogens that have undergone reductive evolution, C. trachomatis is capable of de novo biosynthesis of the essential cofactor tetrahydrofolate using a noncanonical pathway. Here, we identify the biosynthetic precursor to the p-aminobenzoate (pABA) portion of folate in a process that requires the CT610 enzyme from C. trachomatis We further provide evidence that CT610 is a self-sacrificing or "suicide" enzyme that uses its own amino acid residue(s) as the substrate for pABA synthesis. This work provides the foundation for future investigation of this chlamydial pABA synthase, which could lead to new therapeutic strategies for C. trachomatis infections.

Enzymatic and Mutational Analysis of the PruA Pteridine Reductase Required for Pterin-Dependent Control of Biofilm Formation in Agrobacterium tumefaciens.

Pterins are ubiquitous biomolecules with diverse functions including roles as cofactors, pigments, and redox mediators. Recently, a novel pterin-dependent signaling pathway that controls biofilm formation was identified in the plant pathogen, Agrobacterium tumefaciens A key player in this pathway is a pteridine reductase termed PruA, where its enzymatic activity has been shown to control surface attachment and limit biofilm formation. Here, we biochemically characterize PruA to investigate the catalytic properties and substrate specificity of this pteridine reductase. PruA demonstrates maximal catalytic efficiency with dihydrobiopterin and comparable activities with the stereoisomers dihydromonapterin and dihydroneopterin. Since A. tumefaciens does not synthesize or utilize biopterins, the likely physiological substrate is dihydromonapterin or dihydroneopterin, or both. Notably, PruA does not exhibit pteridine reductase activity with dihydrofolate or fully oxidized pterins. Site-directed mutagenesis studies of a conserved tyrosine residue, the key component of a putative catalytic triad, indicate that this tyrosine is not directly involved in PruA catalysis but may be important for substrate or cofactor binding. Additionally, mutagenesis of the arginine residue in the N-terminal TGX3RXG motif significantly reduces the catalytic efficiency of PruA, supporting its proposed role in pterin binding and catalysis. Finally, we report the enzymatic characterization of PruA homologs from Pseudomonas aeruginosa and Brucella abortus, thus expanding the roles and potential significance of pteridine reductases in diverse bacteria.Importance Biofilms are complex multicellular communities that are formed by diverse bacteria. In the plant pathogen, Agrobacterium tumefaciens, the transition from a free-living motile state to a non-motile biofilm state is governed by a novel signaling pathway involving small molecules called pterins. The involvement of pterins in biofilm formation is unexpected and prompts many questions about the molecular details of this pathway. This work biochemically characterizes the PruA pteridine reductase involved in the signaling pathway to reveal its enzymatic properties and substrate preference, thus providing important insight into pterin biosynthesis and its role in A. tumefaciens biofilm control. Additionally, the enzymatic characteristics of related pteridine reductases from mammalian pathogens are examined to uncover potential roles of these enzymes in other bacteria.

Identification and biosynthesis of 2-(1H-imidazol-5-yl) ethan-1-ol (histaminol) in methanogenic archaea.

Histaminol is a relatively rare metabolite most commonly resulting from histidine metabolism. Here we describe histaminol production and secretion into the culture broth by the methanogen Methanococcus maripaludis S2 as well as a number of other methanogens. This work is the first identification of this compound as a natural product in methanogens. Its biosynthesis from histidine was confirmed by the incorporation of 2H3-histidine into histaminol by growing cells of M. maripaludis S2. Possible functions of this molecule could be cell signaling as observed with histamine in eukaryotes or uptake of metal ions.

Identification of a Redox Active Thioquinoxalinol Sulfate Compound Produced by an Anaerobic Methane-Oxidizing Microbial Consortium.

The anaerobic oxidation of methane (AOM) mitigates the flux of methane from marine sediments into the water column. AOM is performed by anaerobic methanotrophic archaea (ANME) that reverse the methanogenesis pathway and partner bacteria that utilize the released reducing equivalents for sulfate reduction. Here, we investigated small-molecule extracts from sediment-free thermophilic enrichment cultures of ANME-1 and sulfate-reducing bacteria using ultraperformance liquid chromatography with high-resolution mass spectrometry. During the analysis, we discovered a novel thioquinoxalinol-containing redox molecule as a major component of the chemically derivatized small-molecule pool. This compound contains both a redox active quinoxaline heterocyclic ring and a thiol group. Additionally, the same core structure was identified that contains a sulfate ester on the hydroxyl group, which likely makes the molecule more water soluble. Hydrated versions of both structures were also observed as major compounds in the extracts. On the basis of reactions of model compounds such as quinoxalin-6-ol, the hydrated version appears to be formed from the addition of water to the dehydropyrazine ring followed by an oxidation. These thioquinoxalinol compounds, which represent completely new structures in biochemistry, may be involved in electron transport processes within and/or between ANME-1 and sulfate-reducing bacteria, may serve protective roles by reacting with toxic compounds such as hydrogen sulfide, or may transport sulfate as a sulfate ester into the sulfate-reducing bacteria.

Identification of the Radical SAM Enzymes Involved in the Biosynthesis of Methanopterin and Coenzyme F420 in Methanogens.

Methanogenic archaea represent a source of unique and fascinating anaerobic biochemistry that includes the involvement of many radical S-adenosyl-l-methionine (SAM) enzymes, some of which have well-established functions, while the majority have currently unknown or only partially understood functions. Here, we describe our strategy for the identification of the radical SAM enzyme that catalyzes the two methylation reactions in methanopterin biosynthesis in Methanocaldococcus jannaschii. Additionally, we describe the similar strategy carried out for the identification of the two radical SAM enzymes required for the biosynthesis of the 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F0) moiety of coenzyme F420 in M. jannaschii. This approach can be employed for future functional identification of radical SAM enzymes with currently unknown functions.

Occurrence and biosynthesis of 3-mercaptopropionic acid in Methanocaldococcus jannaschii.

In a non-targeted analysis of thiol-containing compounds in the hyperthermophilic methanogen Methanocaldococcus jannaschii, we discovered three unexpected metabolites: 3-mercaptopropionic acid (MPA), 2-hydroxy-4-mercaptobutyric acid (HMBA) and 4-mercapto-2-oxobutyric acid (MOB). HMBA and MOB have never been reported as natural products, while MPA is highly prevalent in aquatic environments as a result of biotic and abiotic processing of sulfur-containing compounds. This report provides evidence that HMBA and MOB are part of a biosynthetic pathway to generate MPA in M. jannaschii We show that HMBA can be biosynthesized from malate semialdehyde and hydrogen sulfide, likely using a mechanism similar to that proposed for coenzyme M, coenzyme B and homocysteine biosynthesis in methanogens, where an aldehyde is converted to a thiol. The L-sulfolactate dehydrogenase, derived from the MJ1425 gene, is shown to catalyze the NAD-dependent oxidation of HMBA to MOB. Finally, we demonstrate that HMBA can be used as a biosynthetic precursor to MPA in M. jannaschii cell extracts. This proposed pathway may contribute to the wide occurrence of MPA in marine environments and indicates that MPA must serve some important function in M. jannaschii.

A Pterin-Dependent Signaling Pathway Regulates a Dual-Function Diguanylate Cyclase-Phosphodiesterase Controlling Surface Attachment in Agrobacterium tumefaciens.

UNLABELLED: The motile-to-sessile transition is an important lifestyle switch in diverse bacteria and is often regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). In general, high c-di-GMP concentrations promote attachment to surfaces, whereas cells with low levels of signal remain motile. In the plant pathogen Agrobacterium tumefaciens, c-di-GMP controls attachment and biofilm formation via regulation of a unipolar polysaccharide (UPP) adhesin. The levels of c-di-GMP in A. tumefaciens are controlled in part by the dual-function diguanylate cyclase-phosphodiesterase (DGC-PDE) protein DcpA. In this study, we report that DcpA possesses both c-di-GMP synthesizing and degrading activities in heterologous and native genetic backgrounds, a binary capability that is unusual among GGDEF-EAL domain-containing proteins. DcpA activity is modulated by a pteridine reductase called PruA, with DcpA acting as a PDE in the presence of PruA and a DGC in its absence. PruA enzymatic activity is required for the control of DcpA and through this control, attachment and biofilm formation. Intracellular pterin analysis demonstrates that PruA is responsible for the production of a novel pterin species. In addition, the control of DcpA activity also requires PruR, a protein encoded directly upstream of DcpA with a predicted molybdopterin-binding domain. PruR is hypothesized to be a potential signaling intermediate between PruA and DcpA through an as-yet-unidentified mechanism. This study provides the first prokaryotic example of a pterin-mediated signaling pathway and a new model for the regulation of dual-function DGC-PDE proteins. IMPORTANCE: Pathogenic bacteria often attach to surfaces and form multicellular communities called biofilms. Biofilms are inherently resilient and can be difficult to treat, resisting common antimicrobials. Understanding how bacterial cells transition to the biofilm lifestyle is essential in developing new therapeutic strategies. We have characterized a novel signaling pathway that plays a dominant role in the regulation of biofilm formation in the model pathogen Agrobacterium tumefaciens. This control pathway involves small metabolites called pterins, well studied in eukaryotes, but this is the first example of pterin-dependent signaling in bacteria. The described pathway controls levels of an important intracellular second messenger (cyclic diguanylate monophosphate) that regulates key bacterial processes such as biofilm formation, motility, and virulence. Pterins control the balance of activity for an enzyme that both synthesizes and degrades the second messenger. These findings reveal a complex, multistep pathway that modulates this enzyme, possibly identifying new targets for antibacterial intervention.

Homocysteine is biosynthesized from aspartate semialdehyde and hydrogen sulfide in methanogenic archaea.

The biosynthetic route for homocysteine, intermediate in methionine biosynthesis, is unknown in some methanogenic archaea because homologues of the canonical required genes cannot be identified. Here we demonstrate that Methanocaldococcus jannaschii can biosynthesize homocysteine from aspartate semialdehyde and hydrogen sulfide. Additionally, we confirm the genes involved in this new pathway in Methanosarcina acetivorans. A possible series of reactions in which a thioaldehyde is formed and then reduced to a thiol are proposed. This represents a novel route for the biosynthesis of homocysteine and exemplifies unique aspects of sulfur chemistry occurring in prebiotic environments and in early life forms.

Investigation of enzymatic C-P bond formation using multiple quantum HCP nuclear magnetic resonance spectroscopy.

The biochemical mechanism for the formation of the C-P-C bond sequence found in l-phosphinothricin, a natural product with antibiotic and herbicidal activity, remains unclear. To obtain further insight into the catalytic mechanism of PhpK, the P-methyltransferase responsible for the formation of the second C-P bond in l-phosphinothricin, we utilized a combination of stable isotopes and two-dimensional nuclear magnetic resonance spectroscopy. Exploiting the newly emerged Bruker QCI probe (Bruker Corp.), we specifically designed and ran a (13) C-(31) P multiple quantum (1) H-(13) C-(31) P (HCP) experiment in (1) H-(31) P two-dimensional mode directly on a PhpK-catalyzed reaction mixture using (13) CH3 -labeled methylcobalamin as the methyl group donor. This method is particularly advantageous because minimal sample purification is needed to maximize product visualization. The observed 3:1:1:3 multiplet specifically and unequivocally illustrates direct bond formation between (13) CH3 and (31) P. Related nuclear magnetic resonance experiments based upon these principles may be designed for the study of enzymatic and/or synthetic chemical reaction mechanisms.

Spectroscopic characterization and mechanistic investigation of P-methyl transfer by a radical SAM enzyme from the marine bacterium Shewanella denitrificans OS217.

Natural products containing carbon-phosphorus bonds elicit important bioactivity in many organisms. l-Phosphinothricin contains the only known naturally-occurring carbon-phosphorus-carbon bond linkage. In actinomycetes, the cobalamin-dependent radical S-adenosyl-l-methionine (SAM) methyltransferase PhpK catalyzes the formation of the second C-P bond to generate the complete C-P-C linkage in phosphinothricin. Here we use electron paramagnetic resonance and nuclear magnetic resonance spectroscopies to characterize and demonstrate the activity of a cobalamin-dependent radical SAM methyltransferase denoted SD_1168 from Shewanella denitrificans OS217, a marine bacterium that has not been reported to synthesize phosphinothricin. Recombinant, refolded, and reconstituted SD_1168 binds a four-iron, four-sulfur cluster that interacts with SAM and cobalamin. In the presence of SAM, a reductant, and methylcobalamin, SD_1168 surprisingly catalyzes the P-methylation of N-acetyl-demethylphosphinothricin and demethylphosphinothricin to produce N-acetyl-phosphinothricin and phosphinothricin, respectively. In addition, this enzyme is active in the absence of methylcobalamin if the strong reductant titanium (III) citrate and hydroxocobalamin are provided. When incubated with [methyl-(13)C] cobalamin and titanium citrate, both [methyl-(13)C] and unlabeled N-acetylphosphinothricin are produced. Our results suggest that SD_1168 catalyzes P-methylation using radical SAM-dependent chemistry with cobalamin as a coenzyme. In light of recent genomic information, the discovery of this P-methyltransferase suggests that S. denitrificans produces a phosphinate natural product.