A mixed-valent Fe(II)Fe(III) species converts cysteine to an oxazolone/thioamide pair in methanobactin biosynthesis.

SignificanceMethanobactins (Mbns), copper-binding peptidic compounds produced by some bacteria, are candidate therapeutics for human diseases of copper overload. The paired oxazolone-thioamide bidentate ligands of methanobactins are generated from cysteine residues in a precursor peptide, MbnA, by the MbnBC enzyme complex. MbnBC activity depends on the presence of iron and oxygen, but the catalytically active form has not been identified. Here, we provide evidence that a dinuclear Fe(II)Fe(III) center in MbnB, which is the only representative of a >13,000-member protein family to be characterized, is responsible for this reaction. These findings expand the known roles of diiron enzymes in biology and set the stage for mechanistic understanding, and ultimately engineering, of the MbnBC biosynthetic complex.

Elevated N-Linked Glycosylation of IgG V Regions in Myasthenia Gravis Disease Subtypes.

Elevated N-linked glycosylation of IgG V regions (IgG-VN-Glyc) is an emerging molecular phenotype associated with autoimmune disorders. To test the broader specificity of elevated IgG-VN-Glyc, we studied patients with distinct subtypes of myasthenia gravis (MG), a B cell-mediated autoimmune disease. Our experimental design focused on examining the B cell repertoire and total IgG. It specifically included adaptive immune receptor repertoire sequencing to quantify and characterize N-linked glycosylation sites in the circulating BCR repertoire, proteomics to examine glycosylation patterns of the total circulating IgG, and an exploration of human-derived recombinant autoantibodies, which were studied with mass spectrometry and Ag binding assays to respectively confirm occupation of glycosylation sites and determine whether they alter binding. We found that the frequency of IgG-VN-Glyc motifs was increased in the total BCR repertoire of patients with MG when compared with healthy donors. The elevated frequency was attributed to both biased V gene segment usage and somatic hypermutation. IgG-VN-Glyc could be observed in the total circulating IgG in a subset of patients with MG. Autoantigen binding, by four patient-derived MG autoantigen-specific mAbs with experimentally confirmed presence of IgG-VN-Glyc, was not altered by the glycosylation. Our findings extend prior work on patterns of Ig V region N-linked glycosylation in autoimmunity to MG subtypes.

Development of novel methods for non-canonical myeloma protein analysis with an innovative adaptation of immunofixation electrophoresis, native top-down mass spectrometry, and middle-down de novo sequencing.

OBJECTIVES: Multiple myeloma (MM) is a malignant plasma cell neoplasm, requiring the integration of clinical examination, laboratory and radiological investigations for diagnosis. Detection and isotypic identification of the monoclonal protein(s) and measurement of other relevant biomarkers in serum and urine are pivotal analyses. However, occasionally this approach fails to characterize complex protein signatures. Here we describe the development and application of next generation mass spectrometry (MS) techniques, and a novel adaptation of immunofixation, to interrogate non-canonical monoclonal immunoproteins. METHODS: Immunoprecipitation immunofixation (IP-IFE) was performed on a Sebia Hydrasys Scan2. Middle-down de novo sequencing and native MS were performed with multiple instruments (21T FT-ICR, Q Exactive HF, Orbitrap Fusion Lumos, and Orbitrap Eclipse). Post-acquisition data analysis was performed using Xcalibur Qual Browser, ProSight Lite, and TDValidator. RESULTS: We adapted a novel variation of immunofixation electrophoresis (IFE) with an antibody-specific immunosubtraction step, providing insight into the clonal signature of gamma-zone monoclonal immunoglobulin (M-protein) species. We developed and applied advanced mass spectrometric techniques such as middle-down de novo sequencing to attain in-depth characterization of the primary sequence of an M-protein. Quaternary structures of M-proteins were elucidated by native MS, revealing a previously unprecedented non-covalently associated hetero-tetrameric immunoglobulin. CONCLUSIONS: Next generation proteomic solutions offer great potential for characterizing complex protein structures and may eventually replace current electrophoretic approaches for the identification and quantification of M-proteins. They can also contribute to greater understanding of MM pathogenesis, enabling classification of patients into new subtypes, improved risk stratification and the potential to inform decisions on future personalized treatment modalities.

Novel Interface for High-Throughput Analysis of Biotherapeutics by Electrospray Mass Spectrometry.

With the rapid rise of therapeutic antibodies and antibody-drug conjugates, significant investments have been made in developing workflows that utilize mass spectrometry to detect these intact molecules, the large fragments generated by their selective digestion, and the peptides generated by traditional proteomics workflows. The resultant data is used to gain insight into a wide range of parameters, including primary sequence, disulfide bonding, glycosylation patterns, biotransformation, and more. However, many of the technologies utilized to couple these workflows to mass spectrometers have significant limitations that force nonoptimal modifications to upstream sample preparation steps, limit the throughput of high-volume workflows, and prevent the harmonization of diverse experiments onto a single hardware platform. Here, we describe a new analytical platform that enables direct and high-throughput coupling to electrospray ionization mass spectrometry. The SampleStream platform is compatible with both native and denaturing electrospray, operates with a throughput of up to 15 s/sample, provides extensive concentration of dilute samples, and affords similar sensitivity to comparable liquid chromatographic methods.

Conformational Control of UDP-Galactopyranose Mutase Inhibition.

UDP-galactopyranose mutase (Glf or UGM) catalyzes the formation of uridine 5'-diphosphate-α-d-galactofuranose (UDP-Galf) from UDP-galactopyranose (UDP-Galp). The enzyme is required for the production of Galf-containing glycans. UGM is absent in mammals, but members of the Corynebacterineae suborder require UGM for cell envelope biosynthesis. The need for UGM in some pathogens has prompted the search for inhibitors that could serve as antibiotic leads. Optimizing inhibitor potency, however, has been challenging. The UGM from Klebsiella pneumoniae (KpUGM), which is not required for viability, is more effectively impeded by small-molecule inhibitors than are essential UGMs from species such as Mycobacterium tuberculosis or Corynebacterium diphtheriae. Why KpUGM is more susceptible to inhibition than other orthologs is not clear. One potential source of difference is UGM ortholog conformation. We previously determined a structure of CdUGM bound to a triazolothiadiazine inhibitor in the open form, but it was unclear whether the small-molecule inhibitor bound this form or to the closed form. By varying the terminal tag (CdUGM-His6 and GSG-CdUGM), we crystallized CdUGM to capture the enzyme in different conformations. These structures reveal a pocket in the active site that can be exploited to augment inhibitor affinity. Moreover, they suggest the inhibitor binds the open form of most prokaryotic UGMs but can bind the closed form of KpUGM. This model and the structures suggest strategies for optimizing inhibitor potency by exploiting UGM conformational flexibility.

Steric and electronic effects influencing ß-aryl elimination in the Pd-catalyzed carbon-carbon single bond activation of triarylmethanols.

An analysis of the palladium-catalyzed activation of carbon-carbon single bonds within triarylmethanols has led to a greater understanding of factors influencing the ß-aryl elimination process responsible for C-C bond cleavage. A series of competition reactions were utilized to determine that ß-aryl elimination of aryl substituents containing ortho-substitution proceeds with significant preference to unsubstituted phenyl rings. Further experiments indicate that substrates containing either strongly donating or withdrawing substituents are cleaved from triarylmethanols more readily than relatively neutral species.

Nickel-mediated decarbonylative cross-coupling of phthalimides with in situ generated diorganozinc reagents.

The nickel-mediated cross-coupling of phthalimides with diorganozinc reagents proceeds via a decarbonylative process to produce ortho-substituted benzamides in high yields. In addition to tolerating diverse phthalimide functionality, including alkyl, aryl, and heteroatom containing substituents, this methodology proceeds smoothly with diorganozinc reagents prepared from aryl bromides and utilized without purification.

Chemical composition of material extractives influences microbial growth and dynamics on wetted wood materials.

The impact of material chemical composition on microbial growth on building materials remains relatively poorly understood. We investigate the influence of the chemical composition of material extractives on microbial growth and community dynamics on 30 different wood species that were naturally inoculated, wetted, and held at high humidity for several weeks. Microbial growth was assessed by visual assessment and molecular sequencing. Unwetted material powders and microbial swab samples were analyzed using reverse phase liquid chromatography with tandem mass spectrometry. Different wood species demonstrated varying susceptibility to microbial growth after 3 weeks and visible coverage and fungal qPCR concentrations were correlated (R2 = 0.55). Aspergillaceae was most abundant across all samples; Meruliaceae was more prevalent on 8 materials with the highest visible microbial growth. A larger and more diverse set of compounds was detected from the wood shavings compared to the microbial swabs, indicating a complex and heterogeneous chemical composition within wood types. Several individual compounds putatively identified in wood samples showed statistically significant, near-monotonic associations with microbial growth, including C11H16O4, C18H34O4, and C6H15NO. A pilot experiment confirmed the inhibitory effects of dosing a sample of wood materials with varying concentrations of liquid C6H15NO (assuming it presented as Diethylethanolamine).

Microbial and metabolic succession on common building materials under high humidity conditions.

Despite considerable efforts to characterize the microbial ecology of the built environment, the metabolic mechanisms underpinning microbial colonization and successional dynamics remain unclear, particularly at high moisture conditions. Here, we applied bacterial/viral particle counting, qPCR, amplicon sequencing of the genes encoding 16S and ITS rRNA, and metabolomics to longitudinally characterize the ecological dynamics of four common building materials maintained at high humidity. We varied the natural inoculum provided to each material and wet half of the samples to simulate a potable water leak. Wetted materials had higher growth rates and lower alpha diversity compared to non-wetted materials, and wetting described the majority of the variance in bacterial, fungal, and metabolite structure. Inoculation location was weakly associated with bacterial and fungal beta diversity. Material type influenced bacterial and viral particle abundance and bacterial and metabolic (but not fungal) diversity. Metabolites indicative of microbial activity were identified, and they too differed by material.

Deleterious Consequences of UDP-Galactopyranose Mutase Inhibition for Nematodes.

Parasitic nematodes pose a serious threat to agriculture, livestock, and human health. Increasing resistance to antiparasitic agents underscores the need to replenish our anthelmintic arsenal. The nonpathogenic Caenorhabditis elegans, which serves as an effective model of parasitic helminths, has been used to search for new anthelmintic leads. We previously reported small-molecule inhibitors of the essential C. elegans protein UDP-galactopyranose mutase (UGM or Glf). This enzyme is required for the generation of galactofuranose (Galf)-containing glycans and is needed in nematodes for proper cuticle formation. Though our first-generation inhibitors were effective in vitro, they elicited no phenotypic effects. These findings are consistent with the known difficulty of targeting nematodes. C. elegans is recalcitrant to pharmacological modulation; typically, less than 0.02% of small molecules elicit a phenotypic effect, even at 40 µM. We postulated that the lack of activity of the UGM inhibitors was due to their carboxylic acid group, which can be exploited by nematodes for detoxification. We therefore tested whether replacement of the carboxylate with an N-acylsulfonamide surrogate would result in active compounds. UGM inhibitors with the carboxylate mimetic can phenocopy the deleterious consequences of UGM depletion in C. elegans. These findings support the use of UGM inhibitors as anthelmintic agents. They also outline a strategy to render small-molecule carboxylates more effective against nematodes.

Carboxylate Surrogates Enhance the Antimycobacterial Activity of UDP-Galactopyranose Mutase Probes.

Uridine diphosphate galactopyranose mutase (UGM also known as Glf) is a biosynthetic enzyme required for construction of the galactan, an essential mycobacterial cell envelope polysaccharide. Our group previously identified two distinct classes of UGM inhibitors; each possesses a carboxylate moiety that is crucial for potency yet likely detrimental for cell permeability. To enhance the antimycobacterial potency, we sought to replace the carboxylate with a functional group mimic-an N-acylsulfonamide group. We therefore synthesized a series of N-acylsulfonamide analogs and tested their ability to inhibit UGM. For each inhibitor scaffold tested, the N-acylsulfonamide group functions as an effective carboxylate surrogate. Although the carboxylates and their surrogates show similar activity against UGM in a test tube, several N-acylsulfonamide derivatives more effectively block the growth of Mycobacterium smegmatis. These data suggest that the replacement of a carboxylate with an N-acylsulfonamide group could serve as a general strategy to augment antimycobacterial activity.