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The maturation and installation of the active site metal cluster (FeMo-co, Fe 7 S 9 CMo- R -homocitrate) in Mo-dependent nitrogenase requires the protein product of the nifB gene for production of the FeS cluster precursor (NifB-co, [Fe 8 S 9 C]) and the action of the maturase complex composed of the protein products from the nifE and nifN genes. However, some putative diazotrophic bacteria, like Roseiflexus sp. RS-1, lack the nifEN genes, suggesting an alternative pathway for maturation of FeMo-co that does not require NifEN. In this study, the Roseiflexus NifH, NifB, and apo-NifDK proteins produced in Escherichia coli are shown to be sufficient for FeMo-co maturation and insertion into the NifDK protein to achieve active nitrogenase. The E. coli expressed NifDK RS contained P-clusters but was devoid of FeMo-co (referred to as apo-NifDK RS ). Apo-NifDK RS could be activated for N 2 reduction by addition of preformed FeMo-co. Further, it was found that apo-NifDK RS plus E. coli produced NifB RS and NifH RS were sufficient to yield active NifDK RS when incubated with the necessary substrates (homocitrate, molybdate, and S -adenosylmethionine [SAM]), demonstrating that these proteins can replace the need for NifEN in maturation of Mo-nitrogenase. The E. coli produced NifH RS and NifB RS proteins were independently shown to be functional. The reconstituted NifDK RS demonstrated reduction of N 2 , protons, and acetylene in ratios observed for Azotobacter vinelandii NifDK. These findings reveal a distinct NifEN-independent pathway for nitrogenase activation involving NifH RS , NifB RS , and apo-NifDK RS ., Competing Interests: Competing interests statement:The authors declare no competing interest.

In all domains of life, the biosynthesis of the pterin-based Molybdenum cofactor (Moco) is crucial. Molybdenum (Mo) becomes biologically active by integrating into a unique pyranopterin scaffold, forming Moco. The final two steps of Moco biosynthesis are catalyzed by the two-domain enzyme Mo insertase, linked by gene fusion in higher organisms. Despite well-understood Moco biosynthesis, the evolutionary significance of Mo insertase fusion remains unclear. Here, we present findings from Neurospora crassa that shed light on the critical role of Mo insertase fusion in eukaryotes. Substituting the linkage region with sequences from other species resulted in Moco deficiency, and separate expression of domains, as seen in lower organisms, failed to rescue deficient strains. Stepwise truncation and structural modeling revealed a crucial 20-amino acid sequence within the linkage region essential for fungal growth. Our findings highlight the evolutionary importance of gene fusion and specific sequence composition in eukaryotic Mo insertases., (© 2024. The Author(s).)

Metalloproteins are fundamental to diverse biological processes but still lack extensive investigation in viral contexts. This study reveals the prevalence and functional diversity of metal-binding proteins in DNA viruses. Among a subset of 1432 metalloproteins, zinc and magnesium-binding proteins are notably abundant, indicating their importance in viral biology. Furthermore, significant numbers of proteins binding to iron, manganese, copper, nickel, mercury, and cadmium were also detected. Human-infecting viral proteins displayed a rich landscape of metalloproteins, with MeBiPred (964 proteins) and Pfam (666) yielding the highest numbers. Interestingly, many essential viral proteins exhibited metal-binding capabilities, including polymerases, DNA binding proteins, helicases, dUPTase, thymidine kinase, and various structural and accessory proteins. This study sheds light on the ubiquitous presence of metalloproteins, their functional signatures, subcellular placements, and metal-utilization patterns, providing valuable insights into viral biology. A similar metal utilization pattern was observed in similar functional proteins across the various DNA viruses. Furthermore, these findings provide a foundation for identifying potential drug targets for combating viral infections.

The arsRBC operon encodes a three-protein arsenic resistance system. ArsR regulates the transcription of the operon, while ArsB and ArsC are involved in exporting trivalent arsenic and reducing pentavalent arsenic, respectively. Previous research into Agrobacterium tumefaciens 5A has demonstrated that ArsR has regulatory control over a wide range of metal-related proteins and metabolic pathways. We hypothesized that ArsR has broad regulatory control in other Gram-negative bacteria and set out to test this. Here, we use differential proteomics to investigate changes caused by the presence of the arsR gene in human microbiome-relevant Escherichia coli during arsenite (As III ) exposure. We show that ArsR has broad-ranging impacts such as the expression of TCA cycle enzymes during As III stress. Additionally, we found that the Isc [Fe-S] cluster and molybdenum cofactor assembly proteins are upregulated regardless of the presence of ArsR under these same conditions. An important finding from this differential proteomics analysis was the identification of response mechanisms that were strain-, ArsR-, and arsenic-specific, providing new clarity to this complex regulon. Given the widespread occurrence of the arsRBC operon, these findings should have broad applicability across microbial genera, including sensitive environments such as the human gastrointestinal tract.

Molybdenum cofactor deficiency type A has successfully been treated in a small number of children with daily intravenous administration of cyclic pyranopterin monophosphate. Pharmacodynamic data for this novel treatment have not been published and alternative dosing intervals have not been explored. We monitored pharmacodynamic biomarkers of sulfite oxidase and xanthine oxidoreductase activity in three patients with MoCD-A for a period of 2 to 9 months after discontinuation of cPMP substitution. We found that the clinical and metabolic effects were sustained for longer than expected, over 7 days at least. Our data implicate a biological half-life of the molybdenum cofactor dependent enzyme activities of approximately 3 days and suggest the possibility that less frequent than once daily dosing intervals could be a safe alternative to current practice., Competing Interests: Declaration of competing interest BCS was principal investigator for clinical trials sponsored by Alexion Pharma International, and Origin Biosciences Inc. He reports a personal fee for attendance at an Advisory Board meeting and an unconditional educational grant from Origin Biosciences Inc. KB, HTW, AH, EE and VK have no conflict of interests to report. cPMP for the treatment of patients was provided free of charge on compassionate grounds from Colbourne Pharmaceuticals GmbH and Origin BioSciences Inc., respectively., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Hydrogen-bonding (H-bonding) interactions in metalloprotein active sites can critically regulate enzyme function. Changes in the protein structure triggered by interplay with substrates, products, and partner proteins are often translated to the metallocofactor by way of specific changes in H-bond networks connected to the active site. However, the complexities of metalloprotein architecture and mechanism often preclude our ability to define the precise molecular interactions giving rise to these intricate regulatory pathways. To address this shortcoming, we have developed conformationally switchable artificial metalloproteins (swArMs) in which allosteric Gln-binding triggers protein conformational changes that impact the microenvironment surrounding an installed metallocofactor. Herein, we report a combined structural, spectroscopic, and computational approach to enhance the conformation-dependent changes in H-bond interactions surrounding the metallocofactor site of a swArM. Structure-informed molecular dynamics simulations were employed to predict point mutations that could enhance active site H-bond interactions preferentially in the Gln-bound holo -conformation of the swArM. Testing our predictions via the unique infrared spectral signals associated with the metallocofactor site, we have identified three key residues capable of imparting conformational control over the metallocofactor microenvironment. The resultant swArMs not only model biologically relevant structural regulation but also provide an enhanced Gln-responsive biological probe to be leveraged in future biosensing applications.

Regeneration of insulin-producing β-cells is an alternative avenue to manage diabetes, and it is crucial to unravel this process in vivo during physiological responses to the lack of β-cells. Here, we aimed to characterize how hepatocytes can contribute to β-cell regeneration, either directly or indirectly via secreted proteins or metabolites, in a zebrafish model of β-cell loss. Using lineage tracing, we show that hepatocytes do not directly convert into β-cells even under extreme β-cell ablation conditions. A transcriptomic analysis of isolated hepatocytes after β-cell ablation displayed altered lipid- and glucose-related processes. Based on the transcriptomics, we performed a genetic screen that uncovers a potential role of the molybdenum cofactor (Moco) biosynthetic pathway in β-cell regeneration and glucose metabolism in zebrafish. Consistently, molybdenum cofactor synthesis 2 ( Mocs2 ) haploinsufficiency in mice indicated dysregulated glucose metabolism and liver function. Together, our study sheds light on the liver-pancreas crosstalk and suggests that the molybdenum cofactor biosynthesis pathway should be further studied in relation to glucose metabolism and diabetes., (© 2024 Karampelias et al.)

CRISPR-Cas systems serve as adaptive immune systems in bacteria and archaea, protecting against phages and other mobile genetic elements. However, phages and archaeal viruses have developed countermeasures, employing anti-CRISPR (Acr) proteins to counteract CRISPR-Cas systems. Despite the revolutionary impact of CRISPR-Cas systems on genome editing, concerns persist regarding potential off-target effects. Therefore, understanding the structural and molecular intricacies of diverse Acrs is crucial for elucidating the fundamental mechanisms governing CRISPR-Cas regulation. In this study, we present the structure of AcrIIA28 from Streptococcus phage Javan 128 and analyze its structural and functional features to comprehend the mechanisms involved in its inhibition of Cas9. Our current study reveals that AcrIIA28 is a metalloprotein that contains Zn2+ and abolishes the cleavage activity of Cas9 only from Streptococcus pyrogen (SpyCas9) by directly interacting with the REC3 domain of SpyCas9. Furthermore, we demonstrate that the AcrIIA28 interaction prevents the target DNA from being loaded onto Cas9. These findings indicate the molecular mechanisms underlying AcrIIA28-mediated Cas9 inhibition and provide valuable insights into the ongoing evolutionary battle between bacteria and phages., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Molybdenum cofactor (Moco) biosynthesis is a complex process that involves the coordinated function of several proteins. In the recent years it has become evident that the availability of Fe-S clusters play an important role for the biosynthesis of Moco. First, the MoaA protein binds two [4Fe-4S] clusters per monomer. Second, the expression of the moaABCDE and moeAB operons is regulated by FNR, which senses the availability of oxygen via a functional [4Fe-4S] cluster. Finally, the conversion of cyclic pyranopterin monophosphate to molybdopterin requires the availability of the L-cysteine desulfurase IscS, which is an enzyme involved in the transfer of sulfur to various acceptor proteins with a main role in the assembly of Fe-S clusters. In this review, we dissect the dependence of the production of active molybdoenzymes in detail, starting from the regulation of gene expression and further explaining sulfur delivery and Fe-S cluster insertion into target enzymes. Further, Fe-S cluster assembly is also linked to iron availability. While the abundance of selected molybdoenzymes is largely decreased under iron-limiting conditions, we explain that the expression of the genes is dependent on an active FNR protein. FNR is a very important transcription factor that represents the master-switch for the expression of target genes in response to anaerobiosis. Moco biosynthesis is further directly dependent on the presence of ArcA and also on an active Fur protein., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Silke Leimkuehler reports financial support and administrative support were provided by Deutsche Forschungsgemeinschaft. Silke Leimkuehler reports financial support was provided by German Research Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Molybdenum cofactor sulfurase (MoCoS) is a key gene involved in the uric acid metabolic pathway that activates xanthine dehydrogenase to synthesise uric acid. Uric acid is harmful to mammals but plays crucial roles in insects, one of which is the immune responses. However, the function of Bombyx mori MoCoS in response to BmNPV remains unclear. In this study, BmMoCoS was found to be relatively highly expressed in embryonic development, gonads and the Malpighian tubules. In addition, the expression levels of BmMoCoS were significantly upregulated in three silkworm strains with different levels of resistance after virus infection, suggesting a close link between them. Furthermore, RNAi and overexpression studies showed that BmMoCoS was involved in resistance to BmNPV infection, and its antivirus effects were found to be related to the regulation of uric acid metabolism, which was uncovered by inosine- and febuxostat-coupled RNAi and overexpression. Finally, the BmMoCoS-mediated uric acid pathway was preliminarily confirmed to be a potential target to protect silkworms from BmNPV infection. Overall, this study provides new evidence for elucidating the molecular mechanism of silkworms in response to BmNPV infection and new strategies for the prevention of viral infections in sericulture., (© 2024 Royal Entomological Society.)

Exonic sequences contain both protein-coding and RNA splicing information but the interplay of the protein and splicing code is complex and poorly understood. Here, we have studied traditional and auxiliary splicing codes of human exons that encode residues coordinating two essential divalent metals at the opposite ends of the Irving-Williams series, a universal order of relative stabilities of metal-organic complexes. We show that exons encoding Zn2+-coordinating amino acids are supported much less by the auxiliary splicing motifs than exons coordinating Ca2+. The handicap of the former is compensated by stronger splice sites and uridine-richer polypyrimidine tracts, except for position -3 relative to 3' splice junctions. However, both Ca2+ and Zn2+ exons exhibit close-to-constitutive splicing in multiple tissues, consistent with their critical importance for metalloprotein function and a relatively small fraction of expendable, alternatively spliced exons. These results indicate that constraints imposed by metal coordination spheres on RNA splicing have been efficiently overcome by the plasticity of exon-intron architecture to ensure adequate metalloprotein expression., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The gut microbial metalloenzymes play an important role in maintaining the balance between gut microbial ecosystem, human physiologically processes and immune system. The metals coordinated into active site contribute in various detoxification and defense strategies to avoid unfavourable environment and ensure bacterial survival in human gut. Metallo-β-lactamase is a potent degrader of antibiotics present in periplasmic space of both commensals and pathogenic bacteria. The resistance to anti-microbial agents developed in this enzyme is one of the global threats for human health. The organophosphorus eliminator, organophosphorus hydrolases have evolved over a course of time to hydrolyze toxic organophosphorus compounds and decrease its effect on human health. Further, the redox stress responders namely superoxide dismutase and catalase are key metalloenzymes in reducing both endogenous and exogenous oxidative stress. They hold a great importance for pathogens as they contribute in pathogenesis in human gut along with reduction of oxidative stress. The in-silico study on these enzymes reveals the importance of point mutation for the evolution of these enzymes in order to enhance their enzyme activity and stability. Various mutation studies were conducted to investigate the catalytic activity of these enzymes. By using the "directed evolution" method, the enzymes involved in detoxification and defense system can be engineered to produce new variants with enhance catalytic features, which may be used to predict the severity due to multi-drug resistance and degradation pattern of organophosphorus compounds in human gut., (Copyright © 2024. Published by Elsevier Inc.)

TMEM230 promotes antigen processing, trafficking, and presentation by regulating the endomembrane system of membrane bound organelles (lysosomes, proteosomes and mitochondria) and phagosomes. Activation of the immune system requires trafficking of various cargos between the endomembrane system and cell plasma membrane. The Golgi apparatus is the hub of the endomembrane system and essential for the generation, maintenance, recycling, and trafficking of the components of the endomembrane system itself and immune system. Intracellular trafficking and secretion of immune system components depend on mitochondrial metalloproteins for ATP synthesis that powers motor protein transport of endomembrane cargo. Glycan modifying enzyme genes and motor proteins are essential for the activation of the immune system and trafficking of antigens between the endomembrane system and the plasma membrane. Recently, TMEM230 was identified as co-regulated with RNASET2 in lysosomes and with metalloproteins in various cell types and organelles, including mitochondria in autoimmune diseases. Aberrant metalloproteinase secretion by motor proteins is a major contributor to tissue remodeling of synovial membrane and joint tissue destruction in rheumatoid arthritis (RA) by promoting infiltration of blood vessels, bone erosion, and loss of cartilage by phagocytes. In this study, we identified that specific glycan processing enzymes are upregulated in certain cell types (fibroblast or endothelial cells) that function in destructive tissue remodeling in rheumatoid arthritis compared to osteoarthritis (OA). TMEM230 was identified as a regulator in the secretion of metaloproteinases and heparanase necessary tissue remodeling in OA and RA. In dendritic (DC), natural killer and T cells, TMEM230 was expressed at low or no levels in RA compared to OA. TMEM230 expression in DC likely is necessary for regulatory or helper T cells to maintain tolerance to self-antigens and prevent susceptibility to autoimmune disease. To identify how TMEM230 and the endomembrane system contribute to autoimmunity we investigated, glycan modifying enzymes, metalloproteinases and motor protein genes co-regulated with or regulated by TMEM230 in synovial tissue by analyzing published single cell transcriptomic datasets from RA patient derived synovial tissue., (Copyright © 2024. Published by Elsevier Inc.)

Hybrid cluster proteins (HCPs) are Fe-S-O cluster-containing metalloenzymes in three distinct classes (class I and II: monomer, III: homodimer), all of which structurally related to homodimeric Ni, Fe-carbon monoxide dehydrogenases (CODHs). Here we show X-ray crystal structure of class III HCP from Methanothermobacter marburgensis (Mm HCP), demonstrating its homodimeric architecture structurally resembles those of CODHs. Also, despite the different architectures of class III and I/II HCPs, [4Fe-4S] and hybrid clusters are found in equivalent positions in all HCPs. Structural comparison of Mm HCP and CODHs unveils some distinct features such as the environments of their homodimeric interfaces and the active site metalloclusters. Furthermore, structural analysis of Mm HCP C67Y and characterization of several Mm HCP variants with a Cys67 mutation reveal the significance of Cys67 in protein structure, metallocluster binding and hydroxylamine reductase activity. Structure-based bioinformatics analysis of HCPs and CODHs provides insights into the structural evolution of the HCP/CODH superfamily., (© 2023. Springer Nature Limited.)

Metal-binding proteins are essential for the vital activities and engage in their roles by acting in concert with metal cations. MbPA (The Metal-binding Protein Atlas) is the most comprehensive resource up to now dedicated to curating metal-binding proteins. Currently, it contains 106,373 entries and 440,187 sites related to 54 metals and 8169 species. Users can view all metal-binding proteins and species-specific proteins in MbPA. There are also metal-proteomics data that quantitatively describes protein expression in different tissues and organs. By analyzing the data of the amino acid residues at the metal-binding site, it is found that about 80% of the metal ions tend to bind to cysteine, aspartic acid, glutamic acid, and histidine. Moreover, we use Diversity Measure to confirm that the diversity of metal-binding is specific in different area of periodic table, and further elucidate the binding modes of 19 transition metals on 20 amino acids. In addition, MbPA also embraces 6855 potential pathogenic mutations related to metalloprotein. The resource is freely available at http://bioinfor.imu.edu.cn/mbpa., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Protein engineering has emerged as a powerful methodology to tailor the properties of proteins. It empowers the design of biohybrid catalysts and materials, thereby enabling the convergence of materials science, chemistry, and medicine. The choice of a protein scaffold is an important factor for performance and potential applications. In the past two decades, we utilized the ferric hydroxamate uptake protein FhuA. FhuA is, from our point of view, a versatile scaffold due to its comparably large cavity and robustness toward temperature as well as organic cosolvents. FhuA is a natural iron transporter located in the outer membrane of Escherichia coli ( E. coli ). Wild-type FhuA consists of 714 amino acids and has a β-barrel structure composed of 22 antiparallel β-sheets, closed by an internal globular "cork" domain (amino acids 1-160). FhuA is robust in a broad pH range and toward organic cosolvents; therefore, we envisioned FhuA to be a suitable platform for various applications in (i) biocatalysis, (ii) materials science, and (iii) the construction of artificial metalloenzymes.(i) Applications in biocatalysis were achieved by removing the globular cork domain (FhuA_Δ1-160), thereby creating a large pore for the passive transport of otherwise difficult-to-import molecules through diffusion. Introducing this FhuA variant into the outer membrane of E. coli facilitates the uptake of substrates for downstream biocatalytic conversion. Furthermore, removing the globular "cork" domain without structural collapse of the ß-barrel protein allowed the use of FhuA as a membrane filter, exhibiting a preference for d-arginine over l-arginine.(ii) FhuA is a transmembrane protein, which makes it attractive to be used for applications in non-natural polymeric membranes. Inserting FhuA into polymer vesicles yielded so-called synthosomes (i.e., catalytic synthetic vesicles in which the transmembrane protein acted as a switchable gate or filter). Our work in this direction enables polymersomes to be used in biocatalysis, DNA recovery, and the controlled (triggered) release of molecules. Furthermore, FhuA can be used as a building block to create protein-polymer conjugates to generate membranes.(iii) Artificial metalloenzymes (ArMs) are formed by incorporating a non-native metal ion or metal complex into a protein. This combines the best of two worlds: the vast reaction and substrate scope of chemocatalysis and the selectivity and evolvability of enzymes. With its large inner diameter, FhuA can harbor (bulky) metal catalysts. Among others, we covalently attached a Grubbs-Hoveyda-type catalyst for olefin metathesis to FhuA. This artificial metathease was then used in various chemical transformations, ranging from polymerizations (ring-opening metathesis polymerization) to enzymatic cascades involving cross-metathesis. Ultimately, we generated a catalytically active membrane by copolymerizing FhuA and pyrrole. The resulting biohybrid material was then equipped with the Grubbs-Hoveyda-type catalyst and used in ring-closing metathesis.The number of reports on FhuA and its various applications indicates that it is a versatile building block to generate hybrid catalysts and materials. We hope that our research will inspire future research efforts at the interface of biotechnology, catalysis, and material science in order to create biohybrid systems that offer smart solutions for current challenges in catalysis, material science, and medicine.

Metalation, the acquisition of metals by proteins, must avoid mis-metalation with tighter binding metals. This is illustrated by four selected proteins that require different metals: all show similar ranked orders of affinity for bioavailable metals, as described in a universal affinity series (the Irving-Williams series). Crucially, cellular protein metalation occurs in competition with other metal binding sites. The strength of this competition defines the intracellular availability of each metal: its magnitude has been estimated by calibrating a cells' set of DNA-binding, metal-sensing, transcriptional regulators. This has established that metal availabilities (as free energies for forming metal complexes) are maintained to the inverse of the universal series. The tightest binding metals are least available. With these availabilities, correct metalation is achieved., (© 2022 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Molybdenum cofactor (Moco) is a prosthetic group necessary for the activity of four unique enzymes, including the essential sulfite oxidase (SUOX-1). Moco is required for life; humans with inactivating mutations in the genes encoding Moco-biosynthetic enzymes display Moco deficiency, a rare and lethal inborn error of metabolism. Despite its importance to human health, little is known about how Moco moves among and between cells, tissues, and organisms. The prevailing view is that cells that require Moco must synthesize Moco de novo. Although, the nematode Caenorhabditis elegans appears to be an exception to this rule and has emerged as a valuable system for understanding fundamental Moco biology. C. elegans has the seemingly unique capacity to both synthesize its own Moco as well as acquire Moco from its microbial diet. However, the relative contribution of Moco from the diet or endogenous synthesis has not been rigorously evaluated or quantified biochemically. We genetically removed dietary or endogenous Moco sources in C. elegans and biochemically determined their impact on animal Moco content and SUOX-1 activity. We demonstrate that dietary Moco deficiency dramatically reduces both animal Moco content and SUOX-1 activity. Furthermore, these biochemical deficiencies have physiological consequences; we show that dietary Moco deficiency alone causes sensitivity to sulfite, the toxic substrate of SUOX-1. Altogether, this work establishes the biochemical consequences of depleting dietary Moco or endogenous Moco synthesis in C. elegans and quantifies the surprising contribution of the diet to maintaining Moco homeostasis in C. elegans., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Phosphine ligands are the most important class of ligands for cross-coupling reactions due to their unique electronic and steric properties. However, metalloproteins generally rely on nitrogen, sulfur, or oxygen ligands. Here, we report the genetic incorporation of P3BF, which contains a biocompatible borane-protected phosphine, into proteins. This step is followed by a straightforward one-pot strategy to perform deboronation and palladium coordination in aqueous and aerobic conditions. The genetically encoded phosphine ligand P3BF should significantly expand our ability to design functional metalloproteins.

The potential for ultrahigh-throughput compartmentalization renders droplet microfluidics an attractive tool for the directed evolution of enzymes. Importantly, it ensures maintenance of the phenotype-genotype linkage, enabling reliable identification of improved mutants. Herein, we report an approach for ultrahigh-throughput screening of an artificial metalloenzyme in double emulsion droplets (DEs) using commercially available fluorescence-activated cell sorters (FACS). This protocol was validated by screening a 400 double-mutant streptavidin library for ruthenium-catalyzed deallylation of an alloc-protected aminocoumarin. The most active variants, identified by next-generation sequencing, were in good agreement with hits obtained using a 96-well plate procedure. These findings pave the way for the systematic implementation of FACS for the directed evolution of (artificial) enzymes and will significantly expand the accessibility of ultrahigh-throughput DE screening protocols., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Migration of methane-rich fluids at submarine cold seeps drives intense microbial activity and precipitation of authigenic carbonates. In this study, we analyzed microbially derived authigenic carbonate samples recently recovered from active gas hydrate mounds on the southwestern slope of the Chukchi Borderlands (CB), western Arctic Ocean. Our main aim was to characterize the distribution patterns of trace elements in carbonate-hosted lipid fractions to assess metalloenzyme requirements of microbes involved in anaerobic oxidation of methane (AOM). We measured stable isotopes, trace elements, lipid biomarkers, and genomic DNA, and results indicate the dominance of AOM-related lipid biomarkers in studied carbonate samples, as well as a predominant occurrence of the anaerobic methanotrophic archaea (ANME)-1. We also report evidence for significant preferential enrichments of various trace elements (Li, Ni, Co, Cu, Zn, and Mo) in the total lipid fractions of CB carbonates, relative to elemental compositions determined for corresponding carbonate fractions, which differ from those previously reported for other seep sites. We hypothesize that trace element enrichments in carbonate-hosted lipid fractions could vary depending on the type of AOM microbial assemblage. Additional work is required to further investigate the mechanisms of lipid-bound trace elements in cold seep carbonates as potential metalloenzymes in AOM., (© 2022. The Author(s).)

Background: The expression of ribosomal protein S27 (RPS27) is upregulated in multiple human malignancies. In thyroid cancer, the expression of RPS27 is associated with patient outcomes. However, the carcinogenic mechanisms of RPS27 and functions of RPS27 in the initiation and progression of thyroid cancer are still not clear., Objectives: To investigate the carcinogenic mechanisms of RPS27 and functions of RPS27 in the initiation and progression of thyroid cancer., Material and Methods: The RPS27 gene was overexpressed in BTH101 cells and the influence on the level of gene expression and alternative splicing (AS) was then analyzed by comparing the transcriptomes of the overexpressing cells with the controls. The procedures included cloning and plasmid construction of RPS27, cell culture and transfection, evaluation of RPS27 overexpression, library preparation and sequencing, RNA-Seq raw data clean and alignment, differentially expressed genes (DEGs) analysis, AS analysis, quantitative real-time polymerase chain reaction (qRT-PCR) validation of DEGs and AS events (ASEs), and functional enrichment analysis., Results: The results demonstrated that RPS27 could selectively regulate the expression of genes associated with autoimmune thyroid disease, inflammatory/immune response and AS of genes associated with TRIF-dependent toll-like receptor signaling pathway and apoptotic process. The genes in question are BMP6, SERPINA3, IL17B, IL1RN, HLA-B, PF4, HLA-DOB, MADCAM1, HLA-DQA1, TPO, HLA-B, HLA-DQA1, HLA-DOB, HLA-C, KRT8, CFLAR, HMGA1, CASP8, CCNH, UBE2D3, and MAPK9, among others., Conclusions: The RPS27 selectively regulated the expression and alternative splicing of genes involved in inflammatory/immune response and TRIF-dependent toll-like receptor signaling pathway, which were tightly associated with the initiation and progression of thyroid cancer. These results extend our knowledge on the molecular functions of RPS27 in thyroid cancer cells and have a potential value in thyroid cancer treatment.

Metals play a critical role in human health and diseases. In recent years, metallomics has been introduced and extensively applied to investigate the distribution, regulation, function, and crosstalk of metal(loid) ions in various physiological and pathological processes. Based on high-throughput multielemental analytical techniques and bioinformatics methods, it is possible to elucidate the correlation between the metabolism and homeostasis of diverse metals and complex diseases, in particular for cancer. This review aims to provide an overview of recent progress made in the application of metallomics in cancer research. We mainly focuses on the studies about metallomic profiling of different human biological samples for several major types of cancer, which reveal distinct and dynamic patterns of metal ion contents and the potential benefits of using such information in the detection and prognosis of these malignancies. Elevated levels of copper appear to be a significant risk factor for various cancers, and each type of cancer has a unique distribution of metals in biofluids, hair/nails, and tumor-affected tissues. Furthermore, associations between genetic variations in representative metalloprotein genes and cancer susceptibility have also been demonstrated. Overall, metallomics not only offers a better understanding of the relationship between metal dyshomeostasis and the development of cancer but also facilitates the discovery of new diagnostic and prognostic markers for cancer translational medicine., (© Oxford University Press 2022.)

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality in developing countries. Tripartite motif-59 (TRIM59) a member of the TRIM ubiquitin ligase family, is a surface molecule that regulates biological processes such as cell proliferation, apoptosis, and tumorigenesis. Previous studies reported that TRIM59 expression was upregulated in human CRC, however, the expression pattern and role of TRIM59 in benign colorectal lesions remain unclear. Sixty patients diagnosed with CRC and 60 patients with benign lesions (Crohn's disease, ulcerative colitis, adenoma, and familial adenomatous polyposis) were recruited to the present study. TRIM59 gene expression was assessed by real-time quantitative polymerase chain reaction. Expression of TRIM59 protein and p-AKT were determined using, enzyme-linked immunoassay while p53 expression was detected by immunohistochemistry. Antioxidant/oxidant role of glutathione (GSH)/malondialdehyde (MDA) were evaluated by colorimetric methods in all of the studied groups. Our results showed upregulated expressions of TRIM59 gene and protein levels in CRC tissues and benign colonic lesions compared to nontumor tissues. Their levels were higher in inflammatory compared to noninflammatory bowel lesions. There were significant interrelations among TRIM59 gene expression, protein levels, tumor, node, metastasis staging, and the presence of metastasis (p < 0.0001). Receiver-operator characteristic curve analyses showed that at the cutoff point of 2.5 TRIM59 mRNA expression can discriminate between CRC cases and benign bowel group (area under the curve [AUC]: 0.639, sensitivity: 86.7%, specificity: 41.7%), and between CRC and controls (AUC: 0.962, sensitivity: 90%, specificity: 91.7%). TRIM59 could be a potential biomarker in the early detection, diagnosis, and treatment of benign colonic lesions and CRC., (© 2022 Wiley Periodicals LLC.)

Metals are essential components in life processes and participate in many important biological processes. Dysregulation of metal homeostasis is correlated with many diseases. Metals are also frequently incorporated into diagnosis and therapeutics. Understanding of metal homeostasis under (patho)physiological conditions and the molecular mechanisms of action of metallodrugs in biological systems has positive impacts on human health. As an emerging interdisciplinary area of research, metalloproteomics involves investigating metal-protein interactions in biological systems at a proteome-wide scale, has received growing attention, and has been implemented into metal-related research. In this review, we summarize the recent advances in metalloproteomics methodologies and applications. We also highlight emerging single-cell metalloproteomics, including time-resolved inductively coupled plasma mass spectrometry, mass cytometry, and secondary ion mass spectrometry. Finally, we discuss future perspectives in metalloproteomics, aiming to attract more original research to develop more advanced methodologies, which could be utilized rapidly by biochemists or biologists to expand our knowledge of how metal functions in biology and medicine.

Zinc (Zn) is an essential micronutrient and cofactor for up to 10% of proteins in living organisms. During Zn limitation, specialized enzymes called metallochaperones are predicted to allocate Zn to specific metalloproteins. This function has been putatively assigned to G3E GTPase COG0523 proteins, yet no Zn metallochaperone has been experimentally identified in any organism. Here, we functionally characterize a family of COG0523 proteins that is conserved across vertebrates. We identify Zn metalloprotease methionine aminopeptidase 1 (METAP1) as a COG0523 client, leading to the redesignation of this group of COG0523 proteins as the Zn-regulated GTPase metalloprotein activator (ZNG1) family. Using biochemical, structural, genetic, and pharmacological approaches across evolutionarily divergent models, including zebrafish and mice, we demonstrate a critical role for ZNG1 proteins in regulating cellular Zn homeostasis. Collectively, these data reveal the existence of a family of Zn metallochaperones and assign ZNG1 an important role for intracellular Zn trafficking., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Molecular modelling applications in metalloenzyme design are still scarce due to a series of challenges. On top of that, the simulations of metal-mediated binding and the identification of catalytic competent geometries require both large conformational exploration and simulation of fine electronic properties. Here, we demonstrate how the incorporation of new tools in multiscale strategies, namely substrate diffusion exploration, allows taking a step further. As a showcase, the enantioselective profiles of the most outstanding variants of an artificial Rh 2 -based cyclopropanase (GSH, HFF and RFY) developed by Lewis and co-workers ( Nat. Commun. , 2015, 6 , 7789 and Nat. Chem. , 2018, 10 , 318-324) have been rationalized. DFT calculations on the free-cofactor-mediated process identify the carbene insertion and the cyclopropanoid formation as crucial events, the latter being the enantiodetermining step, which displays up to 8 competitive orientations easily altered by the protein environment. The key intermediates of the reaction were docked into the protein scaffold showing that some mutated residues have direct interaction with the cofactor and/or the co-substrate. These interactions take the form of a direct coordination of Rh in GSH and HFF and a strong hydrophobic patch with the carbene moiety in RFY. Posterior molecular dynamics sustain that the cofactor induces global re-arrangements of the protein. Finally, massive exploration of substrate diffusion, based on the GPathFinder approach, defines this event as the origin of the enantioselectivity in GSH and RFY. For HFF, fine molecular dockings suggest that it is likely related to local interactions upon diffusion. This work shows how modelling of long-range mutations on the catalytic profiles of metalloenzymes may be unavoidable and software simulating substrate diffusion should be applied.

Copper and iron proteins have a wide range of functions in living organisms. Metal assembly into metalloproteins is a complex process, where mismetalation is detrimental and energy consuming to cells. Under metal deficiency, metal distribution is expected to reach a metalation ranking, prioritizing essential versus dispensable metalloproteins, while avoiding interference with other metals and protecting metal-sensitive processes. In this review, we propose that post-transcriptional modulators of metalloprotein mRNA (ModMeR) are good candidates in metal prioritization under metal-limited conditions. ModMeR target high quota or redundant metalloproteins and, by adjusting their synthesis, ModMeR act as internal metal distribution valves. Inappropriate metalation of ModMeR targets could compete with metal delivery to essential metalloproteins and interfere with metal-sensitive processes, such as chloroplastic photosynthesis and mitochondrial respiration. Regulation of ModMeR targets could increase or decrease the metal flow through interconnected pathways in cellular metal distribution, helping to achieve adequate differential metal requirements. Here, we describe and compare ModMeR that function in response to copper and iron deficiencies. Specifically, we describe copper-miRNAs from Arabidopsis thaliana and diverse iron ModMeR from yeast, mammals, and bacteria under copper and iron deficiencies, as well as the influence of oxidative stress. Putative functions derived from their role as ModMeR are also discussed., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Stellacyanin (SC) is a type I (blue) copper protein, which plays a crucial role in plant growth and stress response. However, the comprehensive analysis and functional research of SCs in the woody plant is still lacking. Here, a total of 74 SCs were collected and identified from Arabidopsis, papaya, grape, rice and poplar. Bioinformatics was used to analyze the gene structure, protein structure and evolutionary relationship of 74 genes, especially 19 SCs in Populus trichocarpa. Based on the RNA-seq data, expression pattern of SCs in poplar under cold, high temperature, drought and salt stress were further analyzed. Subsequently, a key candidate gene PtSC18 that strongly responded to drought stress was screened. Subcellular localization experiment exhibited that PtSC18 was localized in the nucleus and plasma membrane. Overexpression of PtSC18 enhanced drought tolerance of transgenic Arabidopsis by improving water retention and reducing oxidative damage. Measurements of physiological indicators, including chlorophyll, H 2 O 2 , malondialdehyde content, peroxidase and catalase enzyme activities and electrical conductivity, all supported this conclusion. More importantly, PtSC18 enhanced the expression of some stress-related genes in transgenic Arabidopsis. Overall, our results lay a foundation for understanding the structure and function of PtSCs and provide useful gene resources for breeding through genetic engineering., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Artificial metalloenzymes (ArMs), created by introducing synthetic cofactors into protein scaffolds, are an emerging class of catalyst for non-natural reactions. Although many classes of ArMs are known, in vitro reconstitution of cofactors and proteins has been a limiting step in the high-throughput screening and directed evolution of ArMs because purification of individual host proteins is time-consuming. We describe the application of a platform to combine mutants of the P450 enzyme CYP119 and the cofactor Ir(Me)MPIX in vivo, by coexpression of the CYP119 mutants with the heme transporter encoded by the hug operon, to the directed evolution of ArMs containing Ir(Me)MPIX in whole cells. We applied this platform to the development an ArMs catalyzing the insertion of the acyclic carbene from α-diazopropanoate esters (Me-EDA) into the N-H bonds of N-alkyl anilines, a combination of carbene and amine classes for which mutant enzymes of natural hemoproteins previously reacted with low enantioselectivity. The mutants of the artificial metalloenzyme Ir(Me)CYP119 identified by an evolution campaign involving more than 4000 mutants are shown to catalyze the reaction of Me-EDA with N-methyl anilines to form chiral chiral amino esters with high TON and good enantioselectivity, thereby demonstrating that the directed evolution of ArMs can rival that of natural enzymes in vivo., (© 2021 Wiley-VCH GmbH.)

The potential applications afforded by the generation and reactivity of artificial metalloenzymes (ArMs) in microorganisms are vast. We show that a non-pathogenic E. coli strain, Nissle 1917 (EcN), is a suitable host for the creation of ArMs from cytochrome P450s and artificial heme cofactors. An outer-membrane receptor in EcN transports an iridium porphyrin into the cell, and the Ir-CYP119 (CYP119 containing iridium porphyrin) assembled in vivo catalyzes carbene insertions into benzylic C-H bonds enantioselectively and site-selectively. The application of EcN as a whole-cell screening platform eliminates the need for laborious processing procedures, drastically increases the ease and throughput of screening, and accelerates the development of Ir-CYP119 with improved catalytic properties. Studies to identify the transport machinery suggest that a transporter different from the previously assumed ChuA receptor serves to usher the iridium porphyrin into the cytoplasm.

Trace elements are required by all organisms, which are key components of many enzymes catalyzing important biological reactions. Many trace element-dependent proteins have been characterized; however, little is known about their occurrence in microbial communities in diverse environments, especially the global marine ecosystem. Moreover, the relationships between trace element utilization and different types of environmental stressors are unclear. In this study, we used metagenomic data from the Global Ocean Sampling expedition project to identify the biogeographic distribution of genes encoding trace element-dependent proteins (for copper, molybdenum, cobalt, nickel, and selenium) in a variety of marine and non-marine aquatic samples. More than 56,000 metalloprotein and selenoprotein genes corresponding to nearly 100 families were predicted, becoming the largest dataset of marine metalloprotein and selenoprotein genes reported to date. In addition, samples with enriched or depleted metalloprotein/selenoprotein genes were identified, suggesting an active or inactive usage of these micronutrients in various sites. Further analysis of interactions among the elements showed significant correlations between some of them, especially those between nickel and selenium/copper. Finally, investigation of the relationships between environmental conditions and metalloprotein/selenoprotein families revealed that many environmental factors might contribute to the evolution of different metalloprotein and/or selenoprotein genes in the marine microbial world. Our data provide new insights into the utilization and biological roles of these trace elements in extant marine microbes, and might also be helpful for the understanding of how these organisms have adapted to their local environments., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Branchio-oto-renal syndrome (BOR) is a disorder characterized by hearing loss, and craniofacial and/or renal defects. Variants in the transcription factor Six1 and its co-factor Eya1, both of which are required for otic development, are linked to BOR. We previously identified Sobp as a potential Six1 co-factor, and SOBP variants in mouse and humans cause otic phenotypes; therefore, we asked whether Sobp interacts with Six1 and thereby may contribute to BOR. Co-immunoprecipitation and immunofluorescence experiments demonstrate that Sobp binds to and colocalizes with Six1 in the cell nucleus. Luciferase assays show that Sobp interferes with the transcriptional activation of Six1+Eya1 target genes. Experiments in Xenopus embryos that either knock down or increase expression of Sobp show that it is required for formation of ectodermal domains at neural plate stages. In addition, altering Sobp levels disrupts otic vesicle development and causes craniofacial cartilage defects. Expression of Xenopus Sobp containing the human variant disrupts the pre-placodal ectoderm similar to full-length Sobp, but other changes are distinct. These results indicate that Sobp modifies Six1 function and is required for vertebrate craniofacial development, and identify Sobp as a potential candidate gene for BOR., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

The epoxide-containing phosphonate natural product fosfomycin is a broad-spectrum antibiotic used in the treatment of cystitis. Fosfomycin is produced by both the plant pathogen Pseudomonas syringae and soil-dwelling streptomycetes. While the streptomycete pathway has recently been fully elucidated, the pseudomonad pathway is still mostly elusive. Through a systematic evaluation of heterologous expression of putative biosynthetic enzymes, we identified the central enzyme responsible for completing the biosynthetic pathway in pseudomonads. The missing transformation involves the oxidative decarboxylation of the intermediate 2-phosphonomethylmalate to a new intermediate, 3-oxo-4-phosphonobutanoate, by PsfC. Crystallographic studies reveal that PsfC unexpectedly belongs to a new class of diiron metalloenzymes that are part of the polymerase and histidinol phosphatase superfamily., Competing Interests: The authors declare no competing interest.

Some methane-oxidizing bacteria use the ribosomally synthesized, posttranslationally modified natural product methanobactin (Mbn) to acquire copper for their primary metabolic enzyme, particulate methane monooxygenase. The operons encoding the machinery to biosynthesize and transport Mbns typically include genes for two proteins, MbnH and MbnP, which are also found as a pair in other genomic contexts related to copper homeostasis. While the MbnH protein, a member of the bacterial diheme cytochrome c peroxidase (bC c P)/MauG superfamily, has been characterized, the structure and function of MbnP, the relationship between the two proteins, and their role in copper homeostasis remain unclear. Biochemical characterization of MbnP from the methanotroph Methylosinus trichosporium OB3b now reveals that MbnP binds a single copper ion, present in the +1 oxidation state, with high affinity. Copper binding to MbnP in vivo is dependent on oxidation of the first tryptophan in a conserved WxW motif to a kynurenine, a transformation that occurs through an interaction of MbnH with MbnP. The 2.04-Å-resolution crystal structure of MbnP reveals a unique fold and an unusual copper-binding site involving a histidine, a methionine, a solvent ligand, and the kynurenine. Although the kynurenine residue may not serve as a Cu I primary-sphere ligand, being positioned ∼2.9 Å away from the Cu I ion, its presence is required for copper binding. Genomic neighborhood analysis indicates that MbnP proteins, and by extension kynurenine-containing copper sites, are widespread and may play diverse roles in microbial copper homeostasis., Competing Interests: The authors declare no competing interest.

The recent pandemic caused by the novel coronavirus resulted in the greatest global health crisis since the Spanish flu pandemic of 1918. There is limited knowledge of whether SARS-CoV-2 is physically associated with human metalloproteins. Recently, high-confidence, experimentally supported protein-protein interactions between SARS-CoV-2 and human proteins were reported. In this work, 58 metalloproteins among these human targets have been identified by a structure-based approach. This study reveals that most human metalloproteins interact with the recently discovered SARS-CoV-2 orf8 protein, whose antibodies are one of the principal markers of SARS-CoV-2 infections. Furthermore, this work provides sufficient evidence to conclude that Zn 2+ plays an important role in the interplay between the novel coronavirus and humans. First, the content of Zn-binding proteins in the involved human metalloproteome is significantly higher than that of the other metal ions. Second, a molecular linkage between the identified human Zn-binding proteome with underlying medical conditions, that might increase the risk of severe illness from the SARS-CoV-2 virus, has been found. Likely perturbations of host cellular metal homeostasis by SARS-CoV-2 infection are highlighted., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Metal cofactors within proteins perform a versatile set of essential cellular functions. In order to take advantage of the diverse functionality of metalloproteins, researchers have been working to design or modify metal binding sites in proteins to rationally tune the function or activity of the metal cofactor. This study has performed an analysis on the backbone atom geometries of metal-binding amino acids among 10 different metal binding sites within the entire protein data bank. A set of 13 geometric parameters (features) was identified that is capable of predicting the presence of a metal cofactor in the protein structure with overall accuracies of up to 97% given only the relative positions of their backbone atoms. The decision tree machine-learning algorithm used can quickly analyze an entire protein structure for the presence of sets of primary metal coordination spheres upon mutagenesis, independent of their original amino acid identities. The methodology was designed for application in the field of metalloprotein engineering. A cluster analysis using the data set was also performed and demonstrated that the features chosen are useful for identifying clusters of structurally similar metal-binding sites., (© 2021 The Protein Society.)

Metalloproteins play key roles across biology, and knowledge of their structure is essential to understand their physiological role. For those metalloproteins containing paramagnetic states, the enhanced relaxation caused by the unpaired electrons often makes signal detection unfeasible near the metal center, precluding adequate structural characterization right where it is more biochemically relevant. Here, we report a protein structure determination by NMR where two different sets of restraints, one containing Nuclear Overhauser Enhancements (NOEs) and another containing Paramagnetic Relaxation Enhancements (PREs), are used separately and eventually together. The protein PioC from Rhodopseudomonas palustris TIE-1 is a High Potential Iron-Sulfur Protein (HiPIP) where the [4Fe-4S] cluster is paramagnetic in both oxidation states at room temperature providing the source of PREs used as alternative distance restraints. Comparison of the family of structures obtained using NOEs only, PREs only, and the combination of both reveals that the pairwise root-mean-square deviation (RMSD) between them is similar and comparable with the precision within each family. This demonstrates that, under favorable conditions in terms of protein size and paramagnetic effects, PREs can efficiently complement and eventually replace NOEs for the structural characterization of small paramagnetic metalloproteins and de novo-designed metalloproteins by NMR. DATABASES: The 20 conformers with the lowest target function constituting the final family obtained using the full set of NMR restraints were deposited to the Protein Data Bank (PDB ID: 6XYV). The 20 conformers with the lowest target function obtained using NOEs only (PDB ID: 7A58) and PREs only (PDB ID: 7A4L) were also deposited to the Protein Data Bank. The chemical shift assignments were deposited to the BMRB (code 34487)., (© 2020 Federation of European Biochemical Societies.)

Artificial metalloenzymes (ArMs) are a class of enzymes holding great promise. In contrast to natural enzymes, the core of ArMs is a synthetic metallocofactor, with potential for bio-orthogonal reactivity, incorporated within a host protein. Next to chemical optimization of the metallocofactor, genetic optimization of the protein allows the further improvement of the ArM. Genetic optimization through directed evolution requires extensive screening of a large sequence-scape to enable the optimization of a desired phenotype. The process is however mostly limited by the throughput of the tools and methods available for screening. In recent years, versatile methods based on droplet microfluidics have been developed to address the need for higher throughput. This article aims to give an introduction into ArMs and the recent technological developments allowing high-throughput directed evolution of enzymes.

Mg-protoporphyrin IX monomethyl ester (MgPME) cyclase catalyses the formation of the isocyclic ring, producing protochlorophyllide a and contributing substantially to the absorption properties of chlorophylls and bacteriochlorophylls. The O 2 -dependent cyclase is found in both oxygenic phototrophs and some purple bacteria. We overproduced the simplest form of the cyclase, AcsF, from Rubrivivax gelatinosus, in Escherichia coli. In biochemical assays the di-iron cluster within AcsF is reduced by ferredoxin furnished by NADPH and ferredoxin:NADP + reductase, or by direct coupling to Photosystem I photochemistry, linking cyclase to the photosynthetic electron transport chain. Kinetic analyses yielded a turnover number of 0.9 min -1 , a Michaelis-Menten constant of 7.0 µM for MgPME and a dissociation constant for MgPME of 0.16 µM. Mass spectrometry identified 13 1 -hydroxy-MgPME and 13 1 -keto-MgPME as cyclase reaction intermediates, revealing the steps that form the isocyclic ring and completing the work originated by Sam Granick in 1950.

Metalloproteins are essential to sustain life. Natural evolution optimized them for intricate structural, regulatory and catalytic functions that cannot be fulfilled by either a protein or a metal ion alone. In order to understand this synergy and the complex design principles behind the natural systems, simpler mimics were engineered from the bottom up by installing de novo metal sites in either natural or fully designed, artificial protein scaffolds. This review focuses on key challenges associated with this approach. We discuss how proteins can be equipped with binding sites that provide an optimal coordination environment for a metal cofactor of choice, which can be a single metal ion or a complex multinuclear cluster. Furthermore, we highlight recent studies in which artificial metalloproteins were engineered towards new functions, including electron transfer and catalysis. In this context, the powerful combination of de novo protein design and directed evolution is emphasized for metalloenzyme development., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Nitrogenase converts N 2 to NH 3 , and CO to hydrocarbons, at its cofactor site. Herein, we report a biochemical and spectroscopic characterization of a Mo-nitrogenase variant expressed in an Azotobacter vinelandii strain containing a deletion of nifV, the gene encoding the homocitrate synthase. Designated NifDK Cit , the catalytic component of this Mo-nitrogenase variant contains a citrate-substituted cofactor analogue. Activity analysis of NifDK Cit reveals a shift of CO reduction from H 2 evolution toward hydrocarbon formation and an opposite shift of N 2 reduction from NH 3 formation toward H 2 evolution. Consistent with a shift in the Mo K-edge energy of NifDK Cit relative to that of its wild-type counterpart, EPR analysis demonstrates a broadening of the line-shape and a decrease in the intensity of the cofactor-originated S=3/2 signal, suggesting a change in the spin properties of the cofactor upon citrate substitution. These observations point to a crucial role of homocitrate in substrate reduction by nitrogenase and the possibility to tune product profiles of nitrogenase reactions via organic ligand substitution., (© 2020 Wiley-VCH GmbH.)

Human aldehyde oxidase (AOX) has emerged as a key enzyme activity for consideration in modern drug discovery. The enzyme catalyzes the oxidation of a wide variety of compounds, most notably azaheterocyclics that often form the building blocks of small molecule therapeutics. Failure to consider and assess AOX drug exposure early in the drug development cycle can have catastrophic consequences for novel compounds entering the clinic. AOX is a complex molybdopterin-containing iron-sulfur flavoprotein comprised of two identical 150 kDa subunits that has proven difficult to produce in recombinant form, and a commercial source of the purified human enzyme is currently unavailable. Thus, the potential exposure of novel drug development candidates to human AOX metabolism is usually assessed by using extracts of pooled human liver cytosol as a source of the enzyme. This can complicate the assignment of AOX-specific compound exposure due to its low activity and the presence of contaminating enzymes that may have overlapping substrate specificities. Herein is described a two-step process for the isolation of recombinant human AOX dimers to near homogeneity following production in the baculovirus expression vector system (BEVS). The deployment of this BEVS-produced recombinant human AOX as a substitute for human liver extracts in a fraction-of-control AOX compound-exposure screening assay is described. The ability to generate this key enzyme activity readily in a purified recombinant form provides for a more accurate and convenient approach to the assessment of new compound exposure to bona fide AOX drug metabolism., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Artificial metalloenzymes, constructed by incorporating a synthetic catalyst into the internal spaces of a protein scaffold, can perform noncanonical chemical transformations that are not possible using natural enzymes. The addition of cell-permeable modules to artificial metalloenzymes allows for noncanonical catalysis to be implemented as a function of mammalian cells. In this chapter, we describe a protocol for controlling cellular function through a cascade consisting of an artificial metalloenzyme and a gene-circuit engineered via synthetic biology.

The molybdenum cofactor (Moco) represents an ancient metal‑sulfur cofactor, which participates as catalyst in carbon, nitrogen and sulfur cycles, both on individual and global scale. Given the diversity of biological processes dependent on Moco and their evolutionary age, Moco is traced back to the last universal common ancestor (LUCA), while Moco biosynthetic genes underwent significant changes through evolution and acquired additional functions. In this review, focused on eukaryotic Moco biology, we elucidate the benefits of gene fusions on Moco biosynthesis and beyond. While originally the gene fusions were driven by biosynthetic advantages such as coordinated expression of functionally related proteins and product/substrate channeling, they also served as origin for the development of novel functions. Today, Moco biosynthetic genes are involved in a multitude of cellular processes and loss of the according gene products result in severe disorders, both related to Moco biosynthesis and secondary enzyme functions., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The molybdenum cofactor (Moco) is a redox active prosthetic group found in the active site of Moco-dependent enzymes (Mo-enzymes). As Moco and its intermediates are highly sensitive towards oxidative damage, these are believed to be permanently protein bound during synthesis and upon maturation. As a major component of the plant Moco transfer and storage system, proteins have been identified that are capable of Moco binding and release but do not possess Moco-dependent enzymatic activities. The first protein found to possess these properties was the Moco carrier protein (MCP) from the green alga Chlamydomonas reinhardtii. Here, we describe the identification and biochemical characterisation of the Volvox carteri (V. carteri) MCP and, for the first time, employ a comparative analysis to elucidate the principles behind MCP Moco binding. Doing so identified a sequence region of low homology amongst the existing MCPs, which we showed to be essential for Moco binding to V. carteri MCP., (© 2020 The Author(s).)

Solute-binding proteins (SBPs) from ATP-binding cassette (ABC) transporters play crucial roles across all forms of life in transporting compounds against chemical gradients. Some SBPs have evolved to scavenge metal substrates from the environment with nanomolar and micromolar affinities (K D ). There exist well established techniques like isothermal titration calorimetry for thoroughly studying these metalloprotein interactions with metal ions, but they are low-throughput. For protein libraries comprised of many metalloprotein homologues and mutants, and for collections of buffer conditions and potential ligands, the throughput of these techniques is paramount. In this study, we describe an improved method termed the microITFQ-LTA and validated it using CjNikZ, a well-characterized nickel-specific SBP (Ni-BP) from Campylobacter jejuni. We then demonstrated how the microITFQ-LTA can be designed to screen through a small collection of buffers and ligands to elucidate the binding profile of a putative Ni-BP from Clostridium carboxidivorans that we call CcSBPII. Through this study, we showed CcSBPII can bind to various metal ions with K D ranged over 3 orders of magnitude. In the presence of l-histidine, CcSBPII could bind to Ni 2+ over 2000-fold more tightly, which was 11.6-fold tighter than CjNikZ given the same ligand., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Recent recurrent outbreaks of Gram-negative bacteria show the critical need to target essential bacterial mechanisms to fight the increase of antibiotic resistance. Pathogenic Gram-negative bacteria have developed several strategies to protect themselves against the host immune response and antibiotics. One such strategy is to remodel the outer membrane where several genes are involved. yejM was discovered as an essential gene in E. coli and S. typhimurium that plays a critical role in their virulence by changing the outer membrane permeability. How the inner membrane protein YejM with its periplasmic domain changes membrane properties remains unknown. Despite overwhelming structural similarity between the periplasmic domains of two YejM homologues with hydrolases like arylsulfatases, no enzymatic activity has been previously reported for YejM. Our studies reveal an intact active site with bound metal ions in the structure of YejM periplasmic domain. Furthermore, we show that YejM has a phosphatase activity that is dependent on the presence of magnesium ions and is linked to its function of regulating outer membrane properties. Understanding the molecular mechanism by which YejM is involved in outer membrane remodeling will help to identify a new drug target in the fight against the increased antibiotic resistance.

Casparian strips (CSs) are cell wall modifications of vascular plants restricting extracellular free diffusion into and out of the vascular system [1]. This barrier plays a critical role in controlling the acquisition of nutrients and water necessary for normal plant development [2-5]. CSs are formed by the precise deposition of a band of lignin approximately 2 μm wide and 150 nm thick spanning the apoplastic space between adjacent endodermal cells [6, 7]. Here, we identified a copper-containing protein, Uclacyanin1 (UCC1), that is sub-compartmentalized within the CS. UCC1 forms a central CS nanodomain in comparison with other CS-located proteins that are found to be mainly accumulated at the periphery of the CS. We found that loss-of-function of two uclacyanins (UCC1 and UCC2) reduces lignification specifically in this central CS nanodomain, revealing a nano-compartmentalized machinery for lignin polymerization. This loss of lignification leads to increased endodermal permeability and, consequently, to a loss of mineral nutrient homeostasis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)