Aminopyrazine Pathway to the Moco Metabolite Dephospho Form A

Klewe, Anne; Kruse, Tobias; Lindel, Thomas

doi:10.1002/chem.201702274

Cited by 11 publications

(12 citation statements)

References 32 publications

(37 reference statements)

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“…The protocol described here was adapted and modified from previous protocols [11,15,19,20] and used synthetic dephospho FormA [21] for Moco/MPT quantification in the biological samples. To quantify Cnx1E bound MPT-AMP and Moco/MPT, the protein preparations were processed directly after elution from the Strep-Tactin ® Superflow ® high-capacity resin (IBA) and prior to concentrating the sample thus ensuring minimal degradation of protein bound Moco/MPT and/or MPT-AMP, respectively.…”

Section: Hplc-based Quantification Of Cnx1e Bound Mpt-amp and Moco/mptmentioning

confidence: 99%

“…All data were collected and processed with OpenLab CDS Version 2.2.0.600. Calibration was carried out using synthetic dephospho FormA [21] as calibration standard. For serial dilution of synthetic dephospho FormA, the protein-free FormA preparation-buffer (see above) was used.…”

Section: Figure 1 Protocol For Forma Based Moco/mpt and Mpt-amp Quanmentioning

confidence: 99%

“…Protein purities of wild-type Cnx1E and Cnx1E variant D274E were found to be equivalent (Supplementary Figure S1), thus allowing their direct comparison with respect to molybdate / MPT-AMP binding and hydrolysis activity. For Moco, metabolite quantification synthetic dephospho FormA [21] ( Figure 3B) may be used as calibration standard for HPLC-based FormA quantification, representing a suitable alternative to the calibration method described earlier [19,20]. Having hands on synthetic dephospho FormA for the HPLC calibration likewise allowed us to establish faster FormA sample preparation protocols (Figures 1 and 3C, see 'Materials and Methods' section for details).…”

Section: Quantitative Analysis Of Cnx1e Variant D274ementioning

confidence: 99%

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Insights into the Cnx1E catalyzed MPT-AMP hydrolysis

et al. 2020

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Molybdenum insertases (Mo-insertases) catalyze the final step of molybdenum cofactor (Moco) biosynthesis, an evolutionary old and highly conserved multi-step pathway. In the first step of the pathway, GTP serves as substrate for the formation of cyclic pyranopterin monophosphate, which is subsequently converted into molybdopterin (MPT) in the second pathway step. In the following synthesis steps, MPT is adenylated yielding MPT-AMP that is subsequently used as substrate for enzyme catalyzed molybdate insertion. Molybdate insertion and MPT-AMP hydrolysis are catalyzed by the Mo-insertase E-domain. Earlier work reported a highly conserved aspartate residue to be essential for Mo-insertase functionality. In this work, we confirmed the mechanistic relevance of this residue for the Arabidopsis thaliana Mo-insertase Cnx1E. We found that the conservative substitution of Cnx1E residue Asp274 by Glu (D274E) leads to an arrest of MPT-AMP hydrolysis and hence to the accumulation of MPT-AMP. We further showed that the MPT-AMP accumulation goes in hand with the accumulation of molybdate. By crystallization and structure determination of the Cnx1E variant D274E, we identified the potential reason for the missing hydrolysis activity in the disorder of the region spanning amino acids 269 to 274. We reasoned that this is caused by the inability of a glutamate in position 274 to coordinate the octahedral Mg2+-water complex in the Cnx1E active site.

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Section: Hplc-based Quantification Of Cnx1e Bound Mpt-amp and Moco/mptmentioning

confidence: 99%

Section: Figure 1 Protocol For Forma Based Moco/mpt and Mpt-amp Quanmentioning

confidence: 99%

Section: Quantitative Analysis Of Cnx1e Variant D274ementioning

confidence: 99%

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Insights into the Cnx1E catalyzed MPT-AMP hydrolysis

et al. 2020

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“…Form A was then deprived of its phosphate group by adding alkaline phosphatase, and the resulting dephospho-Form A was separated in an HPLC setup using a ReproSil-Pur Basic C-18 HD column with dimensions of 250 Â 4.6 mm and 5 mm bead size and was measured with a fluorescence detector. From the area under the curve of the elution peak, the amount of dephospho-Form A in the sample was quantified, with a synthetic Form A standard (Klewe et al, 2017) serving as a reference. The saturation of RoMCP with Moco was calculated as the ratio of the amount of measured dephospho-Form A to the amount of protein subjected to oxidation.…”

Section: Quantification Of Moco Bound To Romcpmentioning

confidence: 99%

The structure of the Moco carrier protein fromRippkaea orientalis

Krausze

Hercher

Archna

et al. 2020

Acta Crystallogr F Struct Biol Commun

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The molybdenum cofactor (Moco) is the prosthetic group of all molybdenum-dependent enzymes except for nitrogenase. The multistep biosynthesis pathway of Moco and its function in molybdenum-dependent enzymes are already well understood. The mechanisms of Moco transfer, storage and insertion, on the other hand, are not. In the cell, Moco is usually not found in its free form and remains bound to proteins because of its sensitivity to oxidation. The green alga Chlamydomonas reinhardtii harbors a Moco carrier protein (MCP) that binds and protects Moco but is devoid of enzymatic function. It has been speculated that this MCP acts as a means of Moco storage and transport. Here, the search for potential MCPs has been extended to the prokaryotes, and many MCPs were found in cyanobacteria. A putative MCP from Rippkaea orientalis (RoMCP) was selected for recombinant production, crystallization and structure determination. RoMCP has a Rossmann-fold topology that is characteristic of nucleotide-binding proteins and a homotetrameric quaternary structure similar to that of the MCP from C. reinhardtii. In each protomer, a positively charged crevice was identified that accommodates up to three chloride ions, hinting at a potential Moco-binding site. Computational docking experiments supported this notion and gave an impression of the RoMCP–Moco complex.

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“…It can be seen that the resin after the ring opening reactions is rich in the hydroxyl groups, leading to abundant hydrogen bonds between the hydroxyl groups and DTPA[AZB] or each other. Meanwhile, the borate bond is a dynamic chemical bond, which makes the π‐π stacking and hydrogen bonding interaction generated by the rigid structure of alizarin tend to be isotropic, thus showing good physicochemical properties …”

Section: Results and Discusstionmentioning

confidence: 99%

A novel nitrogen, phosphorus, and boron ionic pair compound toward fire safety and mechanical enhancement effect for epoxy resin

Tang

Zhou

2019

Polymers for Advanced Techs

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In this work, a novel nitrogen, phosphorus and boron ionic pair compound (DTPA[AZB]), composed of a protonated flame retardant (DTPA) 6,6'‐(1,4‐phenylenebis((pyrazin‐2‐ylamino)methylene))bis(dibenzo[c,e][1,2]oxaphosphinine 6‐oxide) and a counter anion alizarin borate (AZB), has been prepared and fully characterized, AZB was synthesized by the reaction of alizarin with boric acid. DTPA was produced in two steps. First, terephthalaldehyde was condensed with aminopyrazine to form the corresponding imine. This was treated with 9,10‐dihydro‐9‐oxa‐10‐phosphaphenanthrene‐10‐oxide (DOPO) to generate DTPA. Blending with DTPA greatly reduced the flammability of epoxy resin. When the amount of DTPA added was 4%, a modified epoxy resin passed the V‐0 rating and the limiting oxygen index (LOI) reached 32.5%. With the introduction of 3% AZB into the EP/DTPA material, the LOI reached 33.5%. Simultaneously, compared with that of neat EP, the peak heat release rate and smoke production rate for EP/DTPA‐4 was decreased by 24.1% and 40.7%, respectively, and the peak heat release rate and smoke production rate for EP/DTPA[AZB]‐3 was decreased by 32.9% and 43.4%, respectively. The results indicate that AZB and DTPA show good cooperative flame retardant effects. The flame retardancy of the modified epoxy is improved with greater heat release suppression combustion of the resin. A mode of flame retardant action has been proposed based on analysis results from Py‐GC/MS for DTPA, and SEM, IR and Raman for the residual carbon from cone calorimeter and UL‐94 tests, respectively. Importantly, the tensile strength, fexural strength, and fexural modulus of the EP/DTPA[AZB] increased compared with the same properties of neat EP.

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Aminopyrazine Pathway to the Moco Metabolite Dephospho Form A

Cited by 11 publications

References 32 publications

Insights into the Cnx1E catalyzed MPT-AMP hydrolysis

Insights into the Cnx1E catalyzed MPT-AMP hydrolysis

The structure of the Moco carrier protein fromRippkaea orientalis

A novel nitrogen, phosphorus, and boron ionic pair compound toward fire safety and mechanical enhancement effect for epoxy resin

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