Molybdenum Cofactor Biosynthesis Research Articles

Preliminary crystallographic study of the molybdenum cofactor biosynthesis protein MocA complexed with CTP from Mycobacterium sp. strain JC1 DSM 3803

Preliminary crystallographic study of the molybdenum cofactor biosynthesis protein MocA complexed with CTP from <i>Mycobacterium</i> sp. strain JC1 DSM 3803

The molybdenum cofactor biosynthesis gene, OsCNX1, is essential for seedling development and seed germination in rice.

The online version contains supplementary material available at 10.1007/s11032-023-01424-x.

Identification of a Specific Biomarker of Acinetobacter baumannii Global Clone 1 by Machine Learning and PCR Related to Metabolic Fitness of ESKAPE Pathogens.

Since the emergence of high-risk clones worldwide, constant investigations have been undertaken to comprehend the molecular basis that led to their prevalent dissemination in nosocomial settings over time. So far, the complex and multifactorial genetic traits of this type of epidemic clones have allowed only the identification of biomarkers with low specificity. A machine learning algorithm was able to recognize unequivocally a biomarker for early and accurate detection of Acinetobacter baumannii global clone 1 (GC1), one of the most disseminated high-risk clones. A support vector machine model identified the U1 sequence with a length of 367 nucleotides that matched a fragment of the moaCB gene, which encodes the molybdenum cofactor biosynthesis C and B proteins. U1 differentiates specifically between A. baumannii GC1 and non-GC1 strains, becoming a suitable biomarker capable of being translated into clinical settings as a molecular typing method for early diagnosis based on PCR as shown here. Since the metabolic pathways of Mo enzymes have been recognized as putative therapeutic targets for ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens, our findings highlight that machine learning can also be useful in knowledge gaps of high-risk clones and provides noteworthy support to the literature to identify relevant nosocomial biomarkers for other multidrug-resistant high-risk clones. IMPORTANCE A. baumannii GC1 is an important high-risk clone that rapidly develops extreme drug resistance in the nosocomial niche. Furthermore, several strains have been identified worldwide in environmental samples, exacerbating the risk of human interactions. Early diagnosis is mandatory to limit its dissemination and to outline appropriate antibiotic stewardship schedules. A region with a length of 367 bp (U1) within the moaCB gene that is not subjected to lateral genetic transfer or to antibiotic pressures was successfully found by a support vector machine model that predicts A. baumannii GC1 strains. At the same time, research on the group of Mo enzymes proposed this metabolic pathway related to the superbug's metabolism as a potential future drug target site for ESKAPE pathogens due to its central role in bacterial fitness during infection. These findings confirm that machine learning used for the identification of biomarkers of high-risk lineages can also serve to identify putative novel therapeutic target sites.

A step into the rare biosphere: genomic features of the new genus Terrihalobacillus and the new species Aquibacillus salsiterrae from hypersaline soils.

Hypersaline soils are a source of prokaryotic diversity that has been overlooked until very recently. The phylum Bacillota, which includes the genus Aquibacillus, is one of the 26 phyla that inhabit the heavy metal contaminated soils of the Odiel Saltmarshers Natural Area (Southwest Spain), according to previous research. In this study, we isolated a total of 32 strains closely related to the genus Aquibacillus by the traditional dilution-plating technique. Phylogenetic studies clustered them into two groups, and comparative genomic analyses revealed that one of them represents a new species within the genus Aquibacillus, whereas the other cluster constitutes a novel genus of the family Bacillaceae. We propose the designations Aquibacillus salsiterrae sp. nov. and Terrihalobacillus insolitus gen. nov., sp. nov., respectively, for these two new taxa. Genome mining analysis revealed dissimilitude in the metabolic traits of the isolates and their closest related genera, remarkably the distinctive presence of the well-conserved pathway for the biosynthesis of molybdenum cofactor in the species of the genera Aquibacillus and Terrihalobacillus, along with genes that encode molybdoenzymes and molybdate transporters, scarcely found in metagenomic dataset from this area. In-silico studies of the osmoregulatory strategy revealed a salt-out mechanism in the new species, which harbor the genes for biosynthesis and transport of the compatible solutes ectoine and glycine betaine. Comparative genomics showed genes related to heavy metal resistance, which seem required due to the contamination in the sampling area. The low values in the genome recruitment analysis indicate that the new species of the two genera, Terrihalobacillus and Aquibacillus, belong to the rare biosphere of representative hypersaline environments.

MoaB1 Homologs Contribute to Biofilm Formation and Motility by Pseudomonas aeruginosa and Escherichia coli.

moaB homologs, encoding the molybdopterin biosynthetic protein B1, have been reported to be expressed under anoxic conditions and during biofilm growth in various microorganisms; however, little is known about MoaB's function. Here, we demonstrate that in Pseudomonas aeruginosa, MoaB1 (PA3915) contributes to biofilm-related phenotypes. Specifically, moaB1 expression is induced in biofilms, and insertional inactivation of moaB1 reduced biofilm biomass accumulation and pyocyanin production while enhancing swarming motility, and pyoverdine abundance without affecting attachment, swimming motility, or c-di-GMP levels. Inactivation of the highly conserved E. coli homolog of moaB1, moaBEc, likewise coincided with reduced biofilm biomass accumulation. In turn, heterologous expression of moaBEc restored biofilm formation and swarming motility by the P. aeruginosa moaB1 mutant to wild-type levels. Moreover, MoaB1 was found to interact with other conserved biofilm-associated proteins, PA2184 and PA2146, as well as the sensor-kinase SagS. However, despite the interaction, MoaB1 failed to restore SagS-dependent expression of brlR encoding the transcriptional regulator BrlR, and inactivation of moaB1 or moaBEc had no effect on the antibiotic susceptibility phenotype of biofilms formed by P. aeruginosa and E. coli, respectively. While our findings did not establish a link between MoaB1 and molybdenum cofactor biosynthesis, they suggest that MoaB1 homologs contribute to biofilm-associated phenotypes across species boundaries, possibly hinting at the existence of a previously undescribed conserved biofilm pathway. IMPORTANCE Proteins contributing to the biogenesis of molybdenum cofactors have been characterized; however, the role of the molybdopterin biosynthetic protein B1 (MoaB1) has remained elusive, and solid evidence to support its role in biosynthesis of molybdenum cofactor is lacking. Here, we demonstrate that, in Pseudomonas aeruginosa, MoaB1 (PA3915) contributes to biofilm-related phenotypes in a manner that does not support a role of MoaB1 in the biosynthesis of molybdenum cofactors.

Rhodanese-Fold Containing Proteins in Humans: Not Just Key Players in Sulfur Trafficking.

The Rhodanese-fold is a ubiquitous structural domain present in various protein subfamilies associated with different physiological functions or pathophysiological conditions in humans. Proteins harboring a Rhodanese domain are diverse in terms of domain architecture, with some representatives exhibiting one or several Rhodanese domains, fused or not to other structural domains. The most famous Rhodanese domains are catalytically active, thanks to an active-site loop containing an essential cysteine residue which allows for catalyzing sulfur transfer reactions involved in sulfur trafficking, hydrogen sulfide metabolism, biosynthesis of molybdenum cofactor, thio-modification of tRNAs or protein urmylation. In addition, they also catalyse phosphatase reactions linked to cell cycle regulation, and recent advances proposed a new role into tRNA hydroxylation, illustrating the catalytic versatility of Rhodanese domain. To date, no exhaustive analysis of Rhodanese containing protein equipment from humans is available. In this review, we focus on structural and biochemical properties of human-active Rhodanese-containing proteins, in order to provide a picture of their established or putative key roles in many essential biological functions.

The Human Mercaptopyruvate Sulfurtransferase TUM1 Is Involved in Moco Biosynthesis, Cytosolic tRNA Thiolation and Cellular Bioenergetics in Human Embryonic Kidney Cells.

Sulfur is an important element that is incorporated into many biomolecules in humans. The incorporation and transfer of sulfur into biomolecules is, however, facilitated by a series of different sulfurtransferases. Among these sulfurtransferases is the human mercaptopyruvate sulfurtransferase (MPST) also designated as tRNA thiouridine modification protein (TUM1). The role of the human TUM1 protein has been suggested in a wide range of physiological processes in the cell among which are but not limited to involvement in Molybdenum cofactor (Moco) biosynthesis, cytosolic tRNA thiolation and generation of H2S as signaling molecule both in mitochondria and the cytosol. Previous interaction studies showed that TUM1 interacts with the L-cysteine desulfurase NFS1 and the Molybdenum cofactor biosynthesis protein 3 (MOCS3). Here, we show the roles of TUM1 in human cells using CRISPR/Cas9 genetically modified Human Embryonic Kidney cells. Here, we show that TUM1 is involved in the sulfur transfer for Molybdenum cofactor synthesis and tRNA thiomodification by spectrophotometric measurement of the activity of sulfite oxidase and liquid chromatography quantification of the level of sulfur-modified tRNA. Further, we show that TUM1 has a role in hydrogen sulfide production and cellular bioenergetics.

Beyond Moco Biosynthesis-Moonlighting Roles of MoaE and MOCS2.

Molybdenum cofactor (Moco) biosynthesis requires iron, copper, and ATP. The Moco-containing enzyme sulfite oxidase catalyzes terminal oxidation in oxidative cysteine catabolism, and another Moco-containing enzyme, xanthine dehydrogenase, functions in purine catabolism. Thus, molybdenum enzymes participate in metabolic pathways that are essential for cellular detoxication and energy dynamics. Studies of the Moco biosynthetic enzymes MoaE (in the Ada2a-containing (ATAC) histone acetyltransferase complex) and MOCS2 have revealed that Moco biosynthesis and molybdenum enzymes align to regulate signaling and metabolism via control of transcription and translation. Disruption of these functions is involved in the onset of dementia and neurodegenerative disease. This review provides an overview of the roles of MoaE and MOCS2 in normal cellular processes and neurodegenerative disease, as well as directions for future research.

Physiological Importance of Molybdate Transporter Family 1 in Feeding the Molybdenum Cofactor Biosynthesis Pathway in Arabidopsis thaliana.

Molybdate uptake and molybdenum cofactor (Moco) biosynthesis were investigated in detail in the last few decades. The present study critically reviews our present knowledge about eukaryotic molybdate transporters (MOT) and focuses on the model plant Arabidopsis thaliana, complementing it with new experiments, filling missing gaps, and clarifying contradictory results in the literature. Two molybdate transporters, MOT1.1 and MOT1.2, are known in Arabidopsis, but their importance for sufficient molybdate supply to Moco biosynthesis remains unclear. For a better understanding of their physiological functions in molybdate homeostasis, we studied the impact of mot1.1 and mot1.2 knock-out mutants, including a double knock-out on molybdate uptake and Moco-dependent enzyme activity, MOT localisation, and protein–protein interactions. The outcome illustrates different physiological roles for Moco biosynthesis: MOT1.1 is plasma membrane located and its function lies in the efficient absorption of molybdate from soil and its distribution throughout the plant. However, MOT1.1 is not involved in leaf cell imports of molybdate and has no interaction with proteins of the Moco biosynthesis complex. In contrast, the tonoplast-localised transporter MOT1.2 exports molybdate stored in the vacuole and makes it available for re-localisation during senescence. It also supplies the Moco biosynthesis complex with molybdate by direct interaction with molybdenum insertase Cnx1 for controlled and safe sequestering.

Resolving the Multidecade-Long Mystery in MoaA Radical SAM Enzyme Reveals New Opportunities to Tackle Human Health Problems.

MoaA is one of the most conserved radical S-adenosyl-l-methionine (SAM) enzymes, and is found in most organisms in all three kingdoms of life. MoaA contributes to the biosynthesis of molybdenum cofactor (Moco), a redox enzyme cofactor used in various enzymes such as purine and sulfur catabolism in humans and anaerobic respiration in bacteria. Unlike many other cofactors, in most organisms, Moco cannot be taken up as a nutrient and requires de novo biosynthesis. Consequently, Moco biosynthesis has been linked to several human health problems, such as human Moco deficiency disease and bacterial infections. Despite the medical and biological significance, the biosynthetic mechanism of Moco’s characteristic pyranopterin structure remained elusive for more than two decades. This transformation requires the actions of the MoaA radical SAM enzyme and another protein, MoaC. Recently, MoaA and MoaC functions were elucidated as a radical SAM GTP 3′,8-cyclase and cyclic pyranopterin monophosphate (cPMP) synthase, respectively. This finding resolved the key mystery in the field and revealed new opportunities in studying the enzymology and chemical biology of MoaA and MoaC to elucidate novel mechanisms in enzyme catalysis or to address unsolved questions in Moco-related human health problems. Here, we summarize the recent progress in the functional and mechanistic studies of MoaA and MoaC and discuss the field’s future directions.

Structural and Biochemical Studies of Bacillus subtilis MobB

The biosynthesis of molybdenum cofactor for redox enzymes is carried out by multiple enzymes in bacteria including MobA and MobB. MobA is known to catalyze the attachment of GMP to molybdopterin to form molybdopterin guanine dinucleotide. MobB is a GTP binding protein that enhances the activity of MobA by forming the MobA:MobB complex. However, the mechanism of activity enhancement by MobB is not well understood. The structure of Bacillus subtilis MobB was determined to 2.4 Å resolution and it showed an elongated homodimer with an extended β-sheet. Bound sulfate ions were observed in the Walker A motifs, indicating a possible phosphate-binding site for GTP molecules. The binding assay showed that the affinity between B. subtilis MobA and MobB increased in the presence of GTP, suggesting a possible role of MobB as an enhancer of MobA activity.

Engineering of molybdenum-cofactor-dependent nitrate assimilation in Yarrowia lipolytica

ABSTRACTEngineering a new metabolic function in a microbial host can be limited by the availability of the relevant cofactor. For instance, in Yarrowia lipolytica, the expression of a functional nitrate reductase is precluded by the absence of molybdenum cofactor (Moco) biosynthesis. In this study, we demonstrated that the Ogataea parapolymorpha Moco biosynthesis pathway combined with the expression of a high affinity molybdate transporter could lead to the synthesis of Moco in Y. lipolytica. The functionality of Moco was demonstrated by expression of an active Moco-dependent nitrate assimilation pathway from the same yeast donor, O. parapolymorpha. In addition to 11 heterologous genes, fast growth on nitrate required adaptive laboratory evolution which, resulted in up to 100-fold increase in nitrate reductase activity and in up to 4-fold increase in growth rate, reaching 0.13h-1. Genome sequencing of evolved isolates revealed the presence of a limited number of non-synonymous mutations or small insertions/deletions in annotated coding sequences. This study that builds up on a previous work establishing Moco synthesis in S. cerevisiae demonstrated that the Moco pathway could be successfully transferred in very distant yeasts and, potentially, to any other genera, which would enable the expression of new enzyme families and expand the nutrient range used by industrial yeasts.

Mechanism of Reduction of an Aminyl Radical Intermediate in the Radical SAM GTP 3',8-Cyclase MoaA.

The diversity of the reactions catalyzed by radical S-adenosyl-l-methionine (SAM) enzymes is achieved at least in part through the variety of mechanisms to quench their radical intermediates. In the SPASM-twitch family, the largest family of radical SAM enzymes, the radical quenching step is thought to involve an electron transfer to or from an auxiliary 4Fe-4S cluster in or adjacent to the active site. However, experimental demonstration of such functions remains limited. As a representative member of this family, MoaA has one radical SAM cluster ([4Fe-4S]RS) and one auxiliary cluster ([4Fe-4S]AUX), and catalyzes a unique 3',8-cyclization of GTP into 3',8-cyclo-7,8-dihydro-GTP (3',8-cH2GTP) in the molybdenum cofactor (Moco) biosynthesis. Here, we report a mechanistic investigation of the radical quenching step in MoaA, a chemically challenging reduction of 3',8-cyclo-GTP-N7 aminyl radical. We first determined the reduction potentials of [4Fe-4S]RS and [4Fe-4S]AUX as -510 mV and -455 mV, respectively, using a combination of protein film voltammogram (PFV) and electron paramagnetic resonance (EPR) spectroscopy. Subsequent Q-band EPR characterization of 5'-deoxyadenosine C4' radical (5'-dA-C4'•) trapped in the active site revealed isotropic exchange interaction (∼260 MHz) between 5'-dA-C4'• and [4Fe-4S]AUX1+, suggesting that [4Fe-4S]AUX is in the reduced (1+) state during the catalysis. Together with density functional theory (DFT) calculation, we propose that the aminyl radical reduction proceeds through a proton-coupled electron transfer (PCET), where [4Fe-4S]AUX serves as an electron donor and R17 residue acts as a proton donor. These results provide detailed mechanistic insights into the radical quenching step of radical SAM enzyme catalysis.

Split-HaloTag imaging assay for sophisticated microscopy of protein–protein interactions in planta

An ever-increasing number of intracellular multi-protein networks have been identified in plant cells. Split-GFP-based protein–protein interaction assays combine the advantages of in vivo interaction studies in a native environment with additional visualization of protein complex localization. Because of their simple protocols, they have become some of the most frequently used methods. However, standard fluorescent proteins present several drawbacks for sophisticated microscopy. With the HaloTag system, these drawbacks can be overcome, as this reporter forms covalent irreversible bonds with synthetic photostable fluorescent ligands. Dyes can be used in adjustable concentrations and are suitable for advanced microscopy methods. Therefore, we have established the Split-HaloTag imaging assay in plants, which is based on the reconstitution of a functional HaloTag protein upon protein–protein interaction and the subsequent covalent binding of an added fluorescent ligand. Its suitability and robustness were demonstrated using a well-characterized interaction as an example of protein–protein interaction at cellular structures: the anchoring of the molybdenum cofactor biosynthesis complex to filamentous actin. In addition, a specific interaction was visualized in a more distinctive manner with subdiffractional polarization microscopy, Airyscan, and structured illumination microscopy to provide examples of sophisticated imaging. Split-GFP and Split-HaloTag can complement one another, as Split-HaloTag represents an alternative option and an addition to the large toolbox of in vivo methods. Therefore, this promising new Split-HaloTag imaging assay provides a unique and sensitive approach for more detailed characterization of protein–protein interactions using specific microscopy techniques, such as 3D imaging, single-molecule tracking, and super-resolution microscopy.

The effect of dietary protein restriction in a case of molybdenum cofactor deficiency with MOCS1 mutation

Molybdenum cofactor deficiency (MoCD) is an autosomal recessive inborn error of metabolism that results from mutations in genes involved in molybdenum cofactor (Moco) biosynthesis. MoCD is characterized clinically by intractable seizures and severe, rapidly progressing neurodegeneration leading to death in early childhood in the majority of known cases. We report on a patient with an unusual late disease onset and mild phenotype, characterized by delayed development and a decline triggered by a febrile illness and a subsequent dystonic movement disorder. Magnetic resonance imaging showed abnormal signal intensities of the bilateral basal ganglia. Blood and urine chemistry tests demonstrated remarkably low serum and urinary uric acid levels. A urine sulfite test was positive. Specific diagnostic workup showed elevated levels of xanthine and hypoxanthine in serum with increased urinary sulfocysteine (SSC) levels. Genetic analysis revealed a homozygous missense mutation at c.1510C > T (p.504R > W) in exon 10 of the MOCS1 in isoform 7 (rs1387934803). At age 1 year 4 months, the patient was placed on a low protein diet to reduce cysteine load and accumulation of sulfite and SCC in tissues. At 3 months after introduction of protein restriction, the urine sulfite test became negative and the urine SCC level was decreased. After starting the protein restriction diet, dystonic movement improved, and the patient's course progressed without regression and seizures. Electroencephalogram findings were remarkably improved. This finding demonstrates that the dietary protein restriction suppresses disease progression in mild cases of MoCD and suggests the effectiveness of dietary therapy in MoCD.

Coordination Number Regulation of Molybdenum Single-Atom Nanozyme Peroxidase-like Specificity

•A class of MoSA–Nx–C nanozymes are designed and implemented•The peroxidase-like specificity is regulated by Mo–Nx coordination numbers•MoSA–N3–C single-atom nanozyme has superior and exclusive peroxidase-like activity•The mechanism of coordination-number-dependent POD-like specificity is elucidated Nanozymes, which are nanomaterials with enzyme-like characteristics, combine the advantages of nanomaterials and biocatalysts. Although the stability and durability of these nanozymes are comparable to those of natural enzymes, unsatisfactory specificity still limits their wide application as alternatives to natural enzymes. Controlling the targeted enzyme-like performance of traditional nanozymes is extremely challenging owing to the intrinsic structural complexity of nanomaterials. Here, we report the theoretical design and experimental realization of a series of heterogeneous molybdenum single-atom nanozymes. Their peroxidase-like specificity is well regulated by the coordination numbers of single Mo sites. A MoSA–N3–C single-atom nanozyme with superior and specific peroxidase-like activity is demonstrated. This work unravels the structure-selectivity relationships and provides an effective strategy for the rational design of targeted nanozymes. Nanozymes are promising alternatives to natural enzymes, but their use remains limited owing to poor specificity. Overcoming this and controlling the targeted enzyme-like performance of traditional nanozymes is extremely challenging due to the intrinsic structural complexity of these systems. We report theoretical design and experimental realization of a series of heterogeneous molybdenum single-atom nanozymes (named MoSA–Nx–C), wherein we find that the peroxidase-like specificity is well regulated by the coordination numbers of single Mo sites. The resulting MoSA–N3–C catalyst shows exclusive peroxidase-like behavior. It achieves this behavior via a homolytic pathway, whereas MoSA–N2–C and MoSA–N4–C catalysts have a different heterolytic pathway. The mechanism of this coordination-number-dependent enzymatic specificity is attributed to geometrical structure differences and orientation relationships of the frontier molecular orbitals toward these MoSA–Nx–C peroxidase mimics. This study demonstrates the rational design of peroxidase-specific nanozymes and precise regulation of their enzymatic properties. Nanozymes are promising alternatives to natural enzymes, but their use remains limited owing to poor specificity. Overcoming this and controlling the targeted enzyme-like performance of traditional nanozymes is extremely challenging due to the intrinsic structural complexity of these systems. We report theoretical design and experimental realization of a series of heterogeneous molybdenum single-atom nanozymes (named MoSA–Nx–C), wherein we find that the peroxidase-like specificity is well regulated by the coordination numbers of single Mo sites. The resulting MoSA–N3–C catalyst shows exclusive peroxidase-like behavior. It achieves this behavior via a homolytic pathway, whereas MoSA–N2–C and MoSA–N4–C catalysts have a different heterolytic pathway. The mechanism of this coordination-number-dependent enzymatic specificity is attributed to geometrical structure differences and orientation relationships of the frontier molecular orbitals toward these MoSA–Nx–C peroxidase mimics. This study demonstrates the rational design of peroxidase-specific nanozymes and precise regulation of their enzymatic properties. Nanozymes are promising alternatives to natural enzymes and combine the advantages of nanomaterials and biocatalysts,1Wu J. Wang X. Wang Q. Lou Z. Li S. Zhu Y. Qin L. 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Gu W. et al.Densely isolated FeN4 sites for peroxidase mimicking.ACS Catal. 2020; 10: 6422-6429Crossref Scopus (52) Google Scholar This progress clearly indicates considerable promise, but SAC nanozymes are still not suitable for OXD-POD-mediated cascade systems owing to side reactions from OXD-like activities.26Wang Y. Zhang Z. Jia G. Zheng L. Zhao J. Cui X. Elucidating the mechanism of the structure-dependent enzymatic activity of Fe-N/C oxidase mimics.Chem. Commun. (Camb). 2019; 55: 5271-5274Crossref PubMed Google Scholar, 27Huang L. Chen J. Gan L. Wang J. Dong S. Single-atom nanozymes.Sci. Adv. 2019; 5: eaav5490Crossref PubMed Scopus (231) Google Scholar, 28Lu X. Gao S. Lin H. Yu L. Han Y. Zhu P. Bao W. Yao H. Chen Y. Shi J. Bioinspired copper single-atom catalysts for tumor parallel catalytic therapy.Adv. Mater. 2020; 32: 2002246Crossref Scopus (57) Google Scholar Thus, it is crucial to take full advantage of the potential for homogeneity and tunability of SACs to obtain exclusive POD-like specificity. Here, bio-inspired by natural enzymes, we observe that molybdenum is an essential element for the molybdenum cofactor (Moco) in nearly 50 enzyme-catalyzed reactions,29Schwarz G. Mendel R.R. Molybdenum cofactor biosynthesis and molybdenum enzymes.Annu. Rev. Plant Biol. 2006; 57: 623-647Crossref PubMed Scopus (235) Google Scholar,30Kisker C. Schindelin H. Rees D.C. Molybdenum-cofactor-containing enzymes: structure and mechanism.Annu. Rev. Biochem. 1997; 66: 233-267Crossref PubMed Scopus (425) Google Scholar and therefore, we target peroxidase-like single-atom molybdenum nanozymes for building an atomic-level structure-specificity relationship through control of coordination numbers. Theoretical computations are used to design a series of heterogeneous single-atomic molybdenum catalysts (MoSA–Nx–C) with different Mo–Nx coordination numbers (x = 2, 3, 4). These are experimentally fabricated as proof-of-concept systems. They show specific POD-mimicking activities. The correlation between the coordination environment of Mo single-atom sites and peroxidase-like specificity controlled at the atomic level is thus clearly demonstrated by these MoSA–Nx–C nanozymes. The results are understood in detail in terms of geometrical structures and molecular orbitals of MoSA–Nx–C catalysts. The resulting MoSA–N3–C single-atom nanozyme has superior and exclusive peroxidase-like activity. It is successfully applied for selective and sensitive analysis of xanthine in human urine samples. The approach demonstrated thus provides a strategy for unraveling the structure-selectivity relationships for the rational design and realization of selective single-atom nanozymes. Density functional theory (DFT) calculations are used to identify the most promising candidate-specific peroxidase mimics in a series of Mo–Nx–C single-atom models.31Kim M.S. Cho S. Joo S.H. Lee J. Kwak S.K. Kim M.I. Lee J. N- and B-codoped graphene: a strong candidate to replace natural peroxidase in sensitive and selective bioassays.ACS Nano. 2019; 13: 4312-4321Crossref PubMed Scopus (78) Google Scholar,32Hu Y. Gao X.J. Zhu Y. Muhammad F. Tan S. Cao W. Lin S. Jin Z. Gao X. Wei H. Nitrogen-doped carbon nanomaterials as highly active and specific peroxidase mimics.Chem. Mater. 2018; 30: 6431-6439Crossref Scopus (124) Google Scholar The work is guided by the idea that coordination number may be the key controlling parameter, and therefore, particular emphasis is placed on the effect of coordination-number-dependent enzymatic specificity. This is based on Mo–N–C catalysts with different Mo–Nx sites (x = 0, 2, 3, 4) (Figure 1A). The POD-like activity and the major OXD-like interference reaction are assessed by calculating the adsorption energy of H2O2 or O2 on the surface of the catalysts.10Li J. Liu W. Wu X. Gao X. Mechanism of pH-switchable peroxidase and catalase-like activities of gold, silver, platinum and palladium.Biomaterials. 2015; 48: 37-44Crossref PubMed Scopus (210) Google Scholar,26Wang Y. Zhang Z. Jia G. Zheng L. Zhao J. Cui X. Elucidating the mechanism of the structure-dependent enzymatic activity of Fe-N/C oxidase mimics.Chem. Commun. (Camb). 2019; 55: 5271-5274Crossref PubMed Google Scholar,33Wang Y. Qi K. Yu S. Jia G. Cheng Z. Zheng L. Wu Q. Bao Q. Wang Q. Zhao J. et al.Revealing the intrinsic peroxidase-like catalytic mechanism of heterogeneous single-atom Co-MoS2.Nano-Micro Lett. 2019; 11: 102Crossref Scopus (45) Google Scholar As displayed in Figure 1B, the adsorption energies of H2O2 molecules to Mo–Nx–C (x = 2, 3, 4) models are higher than those of O2 molecules, whereas the Mo–C3 sample (the Mo–N0–C model) reveals a reverse trend. The difference of adsorption energy values (ΔEads.) between O2 and H2O2 molecule are taken as convenient descriptors for exploring the dominating enzyme-like property and to obtain more insight.10Li J. Liu W. Wu X. Gao X. Mechanism of pH-switchable peroxidase and catalase-like activities of gold, silver, platinum and palladium.Biomaterials. 2015; 48: 37-44Crossref PubMed Scopus (210) Google Scholar,11Shen X. Liu W. Gao X. Lu Z. Wu X. Gao X. Mechanisms of oxidase and superoxide dismutation-like activities of gold, silver, platinum, and palladium, and their alloys: a general way to the activation of molecular oxygen.J. Am. Chem. Soc. 2015; 137: 15882-15891Crossref PubMed Scopus (223) Google Scholar As shown in Figure 1C, the three-nitrogen-coordinated Mo is more energetically favorable for the selective adsorption of H2O2 with a more negative ΔEads. value (–2.85 eV) as compared with Mo–N2–C (–2.41 eV) and Mo–N4–C (–0.74 eV). These results show that the POD-like activity of Mo–N–C samples is closely dependent on the coordination number. This supports our conjecture about the importance of the coordination number. Importantly, the Mo–N3–C single catalyst is identified as the most promising specific POD-like nanozyme candidate. Single-atom Mo catalysts are prepared by pyrolyzing host-guest templates of molybdenum-doped zeolitic imidazolate framework (Mo-ZIF-8) precursors illustrated in Figure 2A.34Li Z. Chen Y. Ji S. Tang Y. Chen W. Li A. Zhao J. Xiong Y. Wu Y. Gong Y. et al.Iridium single-atom catalyst on nitrogen-doped carbon for formic acid oxidation synthesized using a general host-guest strategy.Nat. Chem. 2020; 12: 764-772Crossref PubMed Scopus (132) Google Scholar The MoO2(acac)2 molecule can be in situ encapsulated within ZIF-8 owing to its proper diameter (9.06 Å) between the size of pores (3.4 Å) and the cavities (11.6 Å) of host ZIF-8 structures (Figure S1; Supplemental Information).35Xiong Y. Dong J. Huang Z.Q. Xin P. Chen W. Wang Y. Li Z. Jin Z. Xing W. Zhuang Z. et al.Single-atom Rh/N-doped carbon electrocatalyst for formic acid oxidation.Nat. Nanotechnol. 2020; 15: 390-397Crossref PubMed Scopus (138) Google Scholar,36Ye W. Chen S. Lin Y. Yang L. Chen S. Zheng X. Qi Z. Wang C. Long R. Chen M. et al.Precisely tuning the number of Fe atoms in clusters on N-doped carbon toward acidic oxygen reduction reaction.Chem. 2019; 5: 2865-2878Abstract Full Text Full Text PDF Scopus (126) Google Scholar Scanning electron microscopy (SEM) images and X-ray diffraction (XRD) patterns of as-prepared Mo-ZIF-8 precursors display uniform rhombic dodecahedron configurations and well-defined crystalline structures consistent with the simulated ZIF-8 crystal (Figure S2). This indicates that doping with guest Mo atoms does not affect the host structure.36Ye W. Chen S. Lin Y. Yang L. Chen S. Zheng X. Qi Z. Wang C. Long R. Chen M. et al.Precisely tuning the number of Fe atoms in clusters on N-doped carbon toward acidic oxygen reduction reaction.Chem. 2019; 5: 2865-2878Abstract Full Text Full Text PDF Scopus (126) Google Scholar The guest MoO2(acac)2 molecules confined in the cage are reduced via pyrolytic conversion into Mo-anchored on nitrogen-doped porous carbon substrates.35Xiong Y. Dong J. Huang Z.Q. Xin P. Chen W. Wang Y. Li Z. Jin Z. Xing W. Zhuang Z. et al.Single-atom Rh/N-doped carbon electrocatalyst for formic acid oxidation.Nat. Nanotechnol. 2020; 15: 390-397Crossref PubMed Scopus (138) Google Scholar The host Zn atoms are gradually evaporated away at high pyrolysis temperature. This decomposes the C–N skeletons and assists the formation of Mo–N bonds.34Li Z. Chen Y. Ji S. Tang Y. Chen W. Li A. Zhao J. Xiong Y. Wu Y. Gong Y. et al.Iridium single-atom catalyst on nitrogen-doped carbon for formic acid oxidation synthesized using a general host-guest strategy.Nat. Chem. 2020; 12: 764-772Crossref PubMed Scopus (132) Google Scholar,37Wang X. Chen Z. Zhao X. Yao T. Chen W. You R. Zhao C. Wu G. Wang J. Huang W. et al.Regulation of coordination number over single Co sites: triggering the efficient electroreduction of CO2.Angew. Chem. Int. Ed. Engl. 2018; 57: 1944-1948Crossref PubMed Scopus (502) Google Scholar Thus, three Mo–Nx–C samples with different N contents but similar Mo contents (Table S1) are controllably prepared under 800°C, 900°C, and 1,000°C and named Mo–N4–C, Mo–N3–C, and Mo–N2–C, respectively.38Hai X. Zhao X. Guo N. Yao C. Chen C. Liu W. Du Y. Yan H. Li J. Chen Z. et al.Engineering local and global structures of single Co atoms for a superior oxygen reduction reaction.ACS Catal. 2020; 10: 5862-5870Crossref Scopus (56) Google Scholar,39Gong Y.-N. Jiao L. Qian Y. Pan C.-Y. Zheng L. Cai X. Liu B. Yu S.-H. Jiang H.-L. Regulating the coordination environment of MOF-templated single-atom nickel electrocatalysts for boosting CO2 reduction.Angew. Chem. Int. Ed. Engl. 2020; 59: 2705-2709Crossref PubMed Scopus (174) Google Scholar There are no obvious differences for physicochemical properties of morphology, size, crystallinity, zeta potential, and surface area of three Mo–Nx–C catalysts (Figures S3–S6; Table S2). No obvious bulk-like metallic Mo phases are observed from XRD patterns (Figure 2B), transmission electron microscopy (TEM) images, and selected area electron diffraction (SAED) images (Figure S3) for these three amorphous carbon-based Mo–Nx–C catalysts. Energy-dispersive X-ray spectroscopy (EDS) mapping shows the homogeneous distribution of C, N, and Mo over the whole nanostructure in three products (Figures 2C and S7). Aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) analysis further provides a direct observation for Mo single atoms as high-density bright dots due to the higher Z-contrast of Mo atoms than N and C atoms (Figures 2D–2F).40Chen W. Pei J. He C.T. Wan J. Ren H. Zhu Y. Wang Y. Dong J. Tian S. Cheong W.-C. et al.Rational design of single molybdenum atoms anchored on N-doped carbon for effective hydrogen evolution reaction.Angew. Chem. Int. Ed. Engl. 2017; 56: 16086-16090Crossref PubMed Scopus (265) Google Scholar,41Zhang L. Jia Y. Gao G. Yan X. Chen N. Chen J. Soo M.T. Wood B. Yang D. Du A. Yao X. Graphene defects trap atomic Ni species for hydrogen and oxygen evolution reactions.Chem. 2018; 4: 285-297Abstract Full Text Full Text PDF Scopus (384) Google Scholar These results demonstrate that single-atom Mo–Nx sites are successfully embedded into the MoSA–Nx–C matrix by the synthesis approach employed. X-ray absorption spectroscopy (XAS) and X-ray photoelectron spectroscopy (XPS) analyses are carried out to explore the coordination environment and electronic information of Mo single atoms. The C and N species are first characterized by Synchrotron-based near-edge X-ray absorption fine structure (NEXAFS). In the C K-edge spectra, three characteristic resonances at about 285.1 eV (π∗ C=C), 288.2 eV (π∗ C–N–C), and 293.0 eV (σ∗ C–C) are identified in all three MoSA–Nx–C catalysts (Figure 3A).42Zheng Y. Jiao Y. Zhu Y. Li L.H. Han Y. Chen Y. Du A. Jaroniec M. Qiao S.Z. Hydrogen evolution by a metal-free electrocatalyst.Nat. Commun. 2014; 5: 3783Crossref PubMed Scopus (1463) Google Scholar From MoSA–N4–C to MoSA–N2–C, the intensity of the π∗ C=C peak gradually increases, whereas the intensity of the σ∗ C–C feature decreases, showing the increased degree of graphitization. Notably, the signal for the π∗ C–N–C bond decreases gradually with the increase of the pyrolysis temperature. This indicates decreased amounts of the C–N–C configuration in the order of MoSA–N4–C, MoSA–N3–C, and MoSA–N2–C, respectively.43Pan Y. Chen Y. Wu K. Chen Z. Liu S. Cao X. Cheong W.C. Meng T. Luo J. Zheng L. et al.Regulating the coordination structure of single-atom Fe-NxCy catalytic sites for benzene oxidation.Nat. Commun. 2019; 10: 4290Crossref PubMed Scopus (116) Google Scholar For the N K-edge spectra, three typical peaks a, b, and c are located at around 398.2, 399.1, and 400.5 eV, corresponding to π∗-transition of pyridinic N, pyrrolic N, and graphitic N species, respectively (Figure 3B).43Pan Y. Chen Y. Wu K. Chen Z. Liu S. Cao X. Cheong W.C. Meng T. Luo J. Zheng L. et al.Regulating the coordination structure of single-atom Fe-NxCy catalytic sites for benzene oxidation.Nat. Commun. 2019; 10: 4290Crossref PubMed Scopus (116) Google Scholar The intensity of the graphitic N species gradually increases, but pyrrolic N and pyridinic N show the opposite trend with the increase of pyrolysis temperature. A similar tendency is also found in the elemental quantification analysis of deconvoluted N species (Figure S8A; Table S3).39Gong Y.-N. Jiao L. Qian Y. Pan C.-Y. Zheng L. Cai X. Liu B. Yu S.-H. Jiang H.-L. Regulating the coordination environment of MOF-templated single-atom nickel electrocatalysts for boosting CO2 reduction.Angew. Chem. Int. Ed. Engl. 2020; 59: 2705-2709Crossref PubMed Scopus (174) Google Scholar According to previous investigations, pyridinic N or pyrrolic N species are capable of donating one p-electron to the π conjugated structure, which are generally considered as coordination sites for atomically dispersed Mo metals.44Zhou H. Zhao Y. Gan J. Xu J. Wang Y. Lv H. Fang S. Wang Z. Deng Z. Wang X. et al.Cation-exchange induced precise regulation of single copper site triggers room-temperature oxidation of benzene.J. Am. Chem. Soc. 2020; 142: 12643-12650Crossref PubMed Scopus (38) Google Scholar Additionally, graphitic N will make a potential impact on the electronic and geometric nanostructures of carbon substrates.45Zhang H. Hwang S. Wang M. Feng Z. Karakalos S. Luo L. Qiao Z. Xie X. Wang C. Su D. et al.Single atomic iron catalysts for oxygen reduction in acidic media: particle size control and thermal activation.J. Am. Chem. Soc. 2017; 139: 14143-14149Crossref PubMed Scopus (738) Google Scholar Consequently, a combination of XAS and XPS results demonstrate that the Mo–N coordination numbers are supposed to be affected and regulated by the pyrolysis temperature. In addition, the evolution of Mo valence states is explored by Mo K-edge X-ray absorption near-edge structure (XANES) (Figure 3C). The absorption edge positions of the three MoSA–Nx–C samples and the Mo-ZIF-8 precursor are situated between those of Mo2C and MoO3 standards, implying that the Mo valence in these catalysts is located between those of the two references.46Tang C. Jiao Y. Shi B. Liu J.N. Xie Z. Chen X. Zhang Q. Qiao S.Z. Coordination tunes selectivity: two-electron oxygen reduction on high-loading molybdenum single-atom catalysts.Angew. Chem. Int. Ed. Engl. 2020; 59: 9171-9176Crossref PubMed Scopus (142) Google Scholar Furthermore, a distinct shift to lower energy values following decreased nitrogen coordination numbers of Mo can be observed. This is in line with the typical trend in the Mo 3D spectra (Figure S8B). Thus, we find that the Mo valence in these MoSA–Nx–C catalysts gradually reduces with increased pyrolysis temperatures.37Wang X. Chen Z. Zhao X. Yao T. Chen W. You R. Zhao C. Wu G. Wang J. Huang W. et al.Regulation of coordination number over single Co sites: triggering the efficient electroreduction of CO2.Angew. Chem. Int. Ed. Engl. 2018; 57: 1944-1948Crossref PubMed Scopus (502) Google Scholar These are calculated from the correlation between Mo valence state and the absorption threshold energy (E0) obtained from the first inflection point in the main adsorption edge region of XANES (Figure S9).40Chen W. Pei J. He C.T. Wan J. Ren H. Zhu Y. Wang Y. Dong J. Tian S. Cheong W.-C. et al.Rational design of single molybdenum atoms anchored on N-doped carbon for effective hydrogen evolution reaction.Angew. Chem. Int. Ed. Engl. 2017; 56: 16086-16090Crossref PubMed Scopus (265) Google Scholar The coordination condition of Mo atoms dispersed in Mo–Nx–C catalysts is explored by the Mo K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy. As shown in Figure 3D, the dominant EXAFS Fourier transform (FT) peaks of these three Mo–Nx–C samples are all around at 1.26 Å, which is ascribed to the Mo–N/O scattering path.40Chen W. Pei J. He C.T. Wan J. Ren H. Zhu Y. Wang Y. Dong J. Tian S. Cheong W.-C. et al.Rational design of single molybdenum atoms anchored on N-doped carbon for effective hydrogen evolution reaction.Angew. Chem. Int. Ed. Engl. 2017; 56: 16086-16090Crossref PubMed Scopus (265) Google Scholar,46Tang C. Jiao Y. Shi B. Liu J.N. Xie Z. Chen X. Zhang Q. Qiao S.Z. Coordination tunes selectivity: two-electron oxygen reduction on high-loading molybdenum single-atom catalysts.Angew. Chem. Int. Ed. Engl. 2020; 59: 9171-9176Crossref PubMed Scopus (142) Google Scholar To further discriminate coordination patterns for the Mo species, wavelet transform (WT) analysis is employed as an ideal supplement for FT.47Funke H. Scheinost A.C. Chukalina M. Wavelet analysis of extended X-ray absorption fine structure data.Phys. Rev. B. 2005; 71: 094110Crossref Scopus (275) Google Scholar,48Shao X.G. Leung A.K.-M. Chau F.T. Wavelet: a new trend in chemistry.Acc. Chem. Res. 2003; 36: 276-283Crossref PubMed Scopus (269) Google Scholar The WT contour plots for all three catalysts possess only one intensity maximum at approximately 5.913 Å−1 assigned to the Mo–N/O coordination (Figure 3E). Moreover, no intensity maximum related to Mo-Mo signals are observed, in contrast with the WT plots of Mo foil, Mo2C, and MoO3. Furthermore, the intensity of Mo–N/O peaks is gradually decreasing with increasing pyrolysis temperature for these MoSA–N4–C, MoSA–N3–C, and MoSA–N2–C samples, suggesting reduced coordination numbers around the atomically dispersed Mo atoms.43Pan Y. Chen Y. Wu K. Chen Z. Liu S. Cao X. Cheong W.C. Meng T. Luo J. Zheng L. et al.Regulating the coordination structure of single-atom Fe-NxCy catalytic sites for benzene oxidation.Nat. Commun. 2019; 10: 4290Crossref PubMed Scopus (116) Google Scholar The coordination number values for these MoSA–Nx–C catalysts are ascertained based on the quantitative EXAFS fitting analysis in k and R spaces. The resulting average Mo–N coordination numbers of Mo within these MoSA–Nx–C samples are 1.8, 3.0, and 4.2, respectively (Figures S10 and S11; Table S4). To further verify the geometrical arrangement of these proposed MoSA–Nx

Genetic analysis of tellurate reduction reveals the selenate/tellurate reductase genes ynfEF and the transcriptional regulation of moeA by NsrR in Escherichia coli.

Several bacteria can reduce tellurate into the less toxic elemental tellurium, but the genes responsible for this process have not yet been identified. In this study, we screened the Keio collection of single-gene knockouts of Escherichia coli responsible for decreased tellurate reduction and found that deletions of 29 genes, including those for molybdenum cofactor (Moco) biosynthesis, iron-sulphur biosynthesis, and the twin-arginine translocation pathway resulted in decreased tellurate reduction. Among the gene knockouts, deletions of nsrR, moeA, yjbB, ynbA, ydaS and yidH affected tellurate reduction more severely than those of other genes. Based on our findings, we determined that the ynfEF genes, which code for the components of the selenate reductase YnfEFGH, are responsible for tellurate reduction. Assays of several molybdoenzymes in the knockouts suggested that nsrR, yjbB, ynbA, ydaS and yidH are essential for the activities of molybdoenzymes in E. coli. Furthermore, we found that the nitric oxide sensor NsrR positively regulated the transcription of the Moco biosynthesis gene moeA. These findings provided new insights into the complexity and regulation of Moco biosynthesis in E. coli.

Molybdenum cofactor biology, evolution and deficiency

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.

Mechanism of Rate Acceleration of Radical C-C Bond Formation Reaction by a Radical SAM GTP 3',8-Cyclase.

While the number of characterized radical S-adenosyl-l-methionine (SAM) enzymes is increasing, the roles of these enzymes in radical catalysis remain largely ambiguous. In radical SAM enzymes, the slow radical initiation step kinetically masks the subsequent steps, making it impossible to study the kinetics of radical chemistry. Due to this kinetic masking, it is unknown whether the subsequent radical reactions require rate acceleration by the enzyme active site. Here, we report the first evidence that a radical SAM enzyme MoaA accelerates the radical-mediated C-C bond formation. MoaA catalyzes an unprecedented 3',8-cyclization of GTP into 3',8-cyclo-7,8-dihydro-GTP (3',8-cH2GTP) during the molybdenum cofactor (Moco) biosynthesis. Through a series of EPR and biochemical characterizations, we found that MoaA catalyzes a shunt pathway in which an on-pathway intermediate, GTP C-3' radical, abstracts H-4' atom from (4'R)-5'-deoxyadenosine (5'-dA) to transiently generate 5'-deoxyadenos-4'-yl radical (5'-dA-C4'•) that is subsequently reduced stereospecifically to yield (4'S)-5'-dA. Detailed kinetic characterization of the shunt and the main pathways provided the comprehensive view of MoaA kinetics and determined the rate of the on-pathway 3',8-cyclization step as 2.7 ± 0.7 s-1. Together with DFT calculations, this observation suggested that the 3',8-cyclization by MoaA is accelerated by 6-9 orders of magnitude. Further experimental and theoretical characterizations suggested that the rate acceleration is achieved mainly by constraining the triphosphate and guanine base positions while leaving the ribose flexible, and a transition state stabilization through H-bond and electrostatic interactions with the positively charged R17 residue. This is the first evidence for rate acceleration of radical reactions by a radical SAM enzyme and provides insights into the mechanism by which radical SAM enzymes accelerate radical chemistry.

The First Step of Neurospora crassa Molybdenum Cofactor Biosynthesis: Regulatory Aspects under N-Derepressing and Nitrate-Inducing Conditions.

Molybdenum cofactor (Moco) is the active site prosthetic group found in all Moco dependent enzymes, except for nitrogenase. Mo-enzymes are crucial for viability throughout all kingdoms of life as they catalyze a diverse set of two electron transfer reactions. The highly conserved Moco biosynthesis pathway consists of four different steps in which guanosine triphosphate is converted into cyclic pyranopterin monophosphate, molybdopterin (MPT), and subsequently adenylated MPT and Moco. Although the enzymes and mechanisms involved in these steps are well characterized, the regulation of eukaryotic Moco biosynthesis is not. Within this work, we described the regulation of Moco biosynthesis in the filamentous fungus Neurospora crassa, which revealed the first step of the multi-step pathway to be under transcriptional control. We found, that upon the induction of high cellular Moco demand a single transcript variant of the nit-7 gene is increasingly formed pointing towards, that essentially the encoded enzyme NIT7-A is the key player for Moco biosynthesis activity in Neurospora.