Discovery of Electrophiles and Profiling of Enzyme Cofactors

AbstractFolate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

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Cofactor Selectivity in Methylmalonyl Coenzyme A Mutase, a Model Cobamide-Dependent Enzyme

mBio ◽

10.1128/mbio.01303-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 6

Author(s):

Olga M. Sokolovskaya ◽

Kenny C. Mok ◽

Jong Duk Park ◽

Jennifer L. A. Tran ◽

Kathryn A. Quanstrom ◽

...

Keyword(s):

Binding Affinity ◽

Bacterial Growth ◽

Microbial Interactions ◽

Vitamin B ◽

Content Type ◽

Domains Of Life ◽

Enzyme Cofactors ◽

Lower Ligand ◽

Enzyme Selectivity

ABSTRACT Cobamides, a uniquely diverse family of enzyme cofactors related to vitamin B12, are produced exclusively by bacteria and archaea but used in all domains of life. While it is widely accepted that cobamide-dependent organisms require specific cobamides for their metabolism, the biochemical mechanisms that make cobamides functionally distinct are largely unknown. Here, we examine the effects of cobamide structural variation on a model cobamide-dependent enzyme, methylmalonyl coenzyme A (CoA) mutase (MCM). The in vitro binding affinity of MCM for cobamides can be dramatically influenced by small changes in the structure of the lower ligand of the cobamide, and binding selectivity differs between bacterial orthologs of MCM. In contrast, variations in the lower ligand have minor effects on MCM catalysis. Bacterial growth assays demonstrate that cobamide requirements of MCM in vitro largely correlate with in vivo cobamide dependence. This result underscores the importance of enzyme selectivity in the cobamide-dependent physiology of bacteria. IMPORTANCE Cobamides, including vitamin B12, are enzyme cofactors used by organisms in all domains of life. Cobamides are structurally diverse, and microbial growth and metabolism vary based on cobamide structure. Understanding cobamide preference in microorganisms is important given that cobamides are widely used and appear to mediate microbial interactions in host-associated and aquatic environments. Until now, the biochemical basis for cobamide preferences was largely unknown. In this study, we analyzed the effects of the structural diversity of cobamides on a model cobamide-dependent enzyme, methylmalonyl-CoA mutase (MCM). We found that very small changes in cobamide structure could dramatically affect the binding affinity of cobamides to MCM. Strikingly, cobamide-dependent growth of a model bacterium, Sinorhizobium meliloti, largely correlated with the cofactor binding selectivity of S. meliloti MCM, emphasizing the importance of cobamide-dependent enzyme selectivity in bacterial growth and cobamide-mediated microbial interactions.

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Versatility in Corrinoid Salvaging and Remodeling Pathways Supports Corrinoid-Dependent Metabolism in Dehalococcoides mccartyi

Applied and Environmental Microbiology ◽

10.1128/aem.02150-12 ◽

2012 ◽

Vol 78 (21) ◽

pp. 7745-7752 ◽

Cited By ~ 77

Author(s):

Shan Yi ◽

Erica C. Seth ◽

Yu-Jie Men ◽

Sally P. Stabler ◽

Robert H. Allen ◽

...

Keyword(s):

De Novo ◽

Chlorinated Solvents ◽

Axial Ligand ◽

Content Type ◽

Dehalococcoides Mccartyi ◽

Axial Ligands ◽

The Common ◽

Enzyme Cofactors ◽

Lower Ligand ◽

Insight Into

ABSTRACTCorrinoids are cobalt-containing molecules that function as enzyme cofactors in a wide variety of organisms but are produced solely by a subset of prokaryotes. Specific corrinoids are identified by the structure of their axial ligands. The lower axial ligand of a corrinoid can be a benzimidazole, purine, or phenolic compound. Though it is known that many organisms obtain corrinoids from the environment, the variety of corrinoids that can serve as cofactors for any one organism is largely unstudied. Here, we examine the range of corrinoids that function as cofactors for corrinoid-dependent metabolism inDehalococcoides mccartyistrain 195.Dehalococcoidesbacteria play an important role in the bioremediation of chlorinated solvents in the environment because of their unique ability to convert the common groundwater contaminants perchloroethene and trichloroethene to the innocuous end product ethene. All isolatedD. mccartyistrains require exogenous corrinoids such as vitamin B12for growth. However, like many other corrinoid-dependent bacteria, none of the well-characterizedD. mccartyistrains has been shown to be capable of synthesizing corrinoidsde novo. In this study, we investigate the ability ofD. mccartyistrain 195 to use specific corrinoids, as well as its ability to modify imported corrinoids to a functional form. We show that strain 195 can use only specific corrinoids containing benzimidazole lower ligands but is capable of remodeling other corrinoids by lower ligand replacement when provided a functional benzimidazole base. This study of corrinoid utilization and modification byD. mccartyiprovides insight into the array of strategies that microorganisms employ in acquiring essential nutrients from the environment.

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Transcriptomic response of an Antarctic yeast Rhodotorula sp. USM-PSY62 to temperature changes

10.1101/2020.05.07.080796 ◽

2020 ◽

Author(s):

Cleo-Nicole Chai ◽

Hok-Chai Yam ◽

Nurlina Rosli ◽

Azali Azlan ◽

Ghows Azzam ◽

...

Keyword(s):

Heat Shock ◽

Protein Transport ◽

Cold Shock ◽

Shock Condition ◽

Transcriptomic Response ◽

Dna And Rna ◽

Antarctic Sea Ice ◽

Magnesium Transporter ◽

Heat Shock Responses ◽

Enzyme Cofactors

AbstractRhodotorula sp. (USM-PSY62) is a psychrophilic yeast isolated from Antarctic sea ice and it grows optimally at 15 °C. This study was set up to observe how USM-PSY62 adapted to fluctuations in temperature. During cold adaptation, an elevated transcription of the CorA magnesium transporter gene in USM-PSY62 indicated a higher requirement for magnesium ions in order to gain additional enzyme cofactors or maintain cytoplasmic fluidity. The HepA homologue coding for DNA/RNA helicase was also over-expressed in cold condition possibly to reorganize secondary structures of DNA and RNA. An up-regulation of the catalase gene was also observed reflecting an increment in the concentration of reactive oxygen species and fluctuations in the associated antioxidant system. The YOP1 gene, which encodes a membrane protein associated with protein transport and membrane traffic, was the most down-regulated under cold shock condition. The genes responsible for the structural maintenance of chromosome (SMC) were also down-regulated when the temperature was shifted to 0 °C. Upon cold shock, the gene for heat shock factor protein 1 (HSF1) was also down-regulated. Hsf1 is a transcriptional regulator which regulate the heat shock responses. Although USM-PSY62 showed some common adaptive strategies as in several other psychrophilic organisms, new mechanisms were also uncovered.

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Manganese influx and expression of ZIP8 is essential in primary myoblasts and contributes to activation of SOD2

10.1101/494542 ◽

2018 ◽

Cited By ~ 2

Author(s):

Shellaina J. V. Gordon ◽

Daniel E. Fenker ◽

Katherine E. Vest ◽

Teresita Padilla-Benavides

Keyword(s):

Skeletal Muscle ◽

Trace Elements ◽

Second Messengers ◽

Muscle Cells ◽

Skeletal Muscle Cells ◽

Metal Transporters ◽

Primary Myoblasts ◽

Zip Family ◽

Enzyme Cofactors ◽

Functional Relevance

ABSTRACTTrace elements such as copper (Cu), zinc (Zn), iron (Fe), and manganese (Mn) are enzyme cofactors and second messengers in cell signaling. Trace elements are emerging as key regulators of differentiation and development of mammalian tissues including blood, brain, and skeletal muscle. We previously reported an influx of Cu and dynamic expression of various metal transporters during differentiation of skeletal muscle cells. Here, we demonstrate that during differentiation of skeletal myoblasts an increase of additional trace elements such as Mn, Fe and Zn occurs. Interestingly the Mn increase is concomitant with increased Mn-dependent SOD2 levels. To better understand the Mn import pathway in skeletal muscle cells, we probed the functional relevance of the closely related proteins ZIP8 and ZIP14, which are implicated in Zn, Mn, and Fe transport. Partial depletion of ZIP8 severely impaired growth of myoblasts and led to cell death under differentiation conditions, indicating that ZIP8-mediated metal transport is essential in skeletal muscle cells. Moreover, knockdown of Zip8 impaired activity of the Mn-dependent SOD2. Growth defects were partially rescued by Mn supplementation to the medium, suggesting additional functions for ZIP8 in the skeletal muscle lineage. Knockdown of Zip14, on the other hand, had only a mild effect on myotube size, consistent with a role for ZIP14 in muscle hypertrophy. This is the first report on the functional relevance of two members of the ZIP family of metal transporters in the skeletal muscle lineage, and further supports the paradigm that trace metal transporters are critical modulators of mammalian tissue development.

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A salvaging strategy enables stable metabolite provisioning among free-living bacteria

10.1101/2021.12.14.472564 ◽

2021 ◽

Author(s):

Sebastian Gude ◽

Gordon J Pherribo ◽

Michiko E Taga

Keyword(s):

De Novo ◽

Metabolic Processes ◽

Free Living ◽

Bacterial Populations ◽

De Novo Biosynthesis ◽

Efficient Route ◽

Related Enzyme ◽

Enzyme Cofactors ◽

Living Bacteria

All organisms rely on complex metabolites such as amino acids, nucleotides, and cofactors for essential metabolic processes. Some microbes synthesize these fundamental ingredients of life de novo, while others rely on uptake to fulfill their metabolic needs. Although certain metabolic processes are inherently 'leaky', the mechanisms enabling stable metabolite provisioning among microbes in the absence of a host remain largely unclear. In particular, how can metabolite provisioning among free-living bacteria be maintained under the evolutionary pressure to economize resources? Salvaging, the process of 'recycling and reusing', can be a metabolically efficient route to obtain access to required resources. Here, we show experimentally how precursor salvaging in engineered Escherichia coli populations can lead to stable, long-term metabolite provisioning. We find that salvaged cobamides (vitamin B12 and related enzyme cofactors) are readily made available to non-productive population members, yet salvagers are strongly protected from overexploitation due to partial metabolite privatization. We also describe a previously unnoted benefit of precursor salvaging, namely the removal of the non-functional, proliferation-inhibiting precursor. As long as compatible precursors are present, any microbe possessing the terminal steps of a biosynthetic process can, in principle, forgo de novo biosynthesis in favor of salvaging. Consequently, precursor salvaging likely represents a potent, yet overlooked, alternative to de novo biosynthesis for the acquisition and provisioning of metabolites in free-living bacterial populations.

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Functional diversity of organic molecule enzyme cofactors

Natural Product Reports ◽

10.1039/c3np70045c ◽

2013 ◽

Vol 30 (10) ◽

pp. 1324 ◽

Cited By ~ 35

Author(s):

Michael Richter

Keyword(s):

Functional Diversity ◽

Organic Molecule ◽

Enzyme Cofactors

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NAD+ metabolism: pathophysiologic mechanisms and therapeutic potential

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-020-00311-7 ◽

2020 ◽

Vol 5 (1) ◽

Author(s):

Na Xie ◽

Lu Zhang ◽

Wei Gao ◽

Canhua Huang ◽

Peter Ernst Huber ◽

...

Keyword(s):

Communication Networks ◽

Molecular Mechanisms ◽

Metabolic Diseases ◽

Environmental Changes ◽

Therapeutic Potential ◽

Nad Metabolism ◽

Oxidation Reduction ◽

Enzyme Cofactors ◽

Genotoxic Factors ◽

Post Synthesis

Abstract Nicotinamide adenine dinucleotide (NAD+) and its metabolites function as critical regulators to maintain physiologic processes, enabling the plastic cells to adapt to environmental changes including nutrient perturbation, genotoxic factors, circadian disorder, infection, inflammation and xenobiotics. These effects are mainly achieved by the driving effect of NAD+ on metabolic pathways as enzyme cofactors transferring hydrogen in oxidation-reduction reactions. Besides, multiple NAD+-dependent enzymes are involved in physiology either by post-synthesis chemical modification of DNA, RNA and proteins, or releasing second messenger cyclic ADP-ribose (cADPR) and NAADP+. Prolonged disequilibrium of NAD+ metabolism disturbs the physiological functions, resulting in diseases including metabolic diseases, cancer, aging and neurodegeneration disorder. In this review, we summarize recent advances in our understanding of the molecular mechanisms of NAD+-regulated physiological responses to stresses, the contribution of NAD+ deficiency to various diseases via manipulating cellular communication networks and the potential new avenues for therapeutic intervention.

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