Insights into the Cnx1E catalyzed MPT-AMP hydrolysis

Hercher, Thomas W.; Krausze, J.; Hoffmeister, Sven; Zwerschke, Dagmar; Lindel, Thomas; Blankenfeldt, Wulf; Mendel, Ralf R.; Kruse, Tobias

doi:10.1042/bsr20191806

Cited by 16 publications

(30 citation statements)

References 34 publications

(81 reference statements)

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“…The quantification of the Moco content in RoMCP samples followed a previously published protocol (Hercher et al, 2020). Briefly, the Moco content of a sample containing 100 pmol RoMCP was oxidized with a solution containing 1% I 2 and 2% KI under acidic conditions and converted to the compound Form A.…”

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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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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“…4). Free Moco is highly unstable, however it can be Warnhoff 6 oxidized to 'Form A', a stable and fluorescent Moco-derivative that is quantifiable via HPLC (Johnson et al 1980;Hercher et al 2020a). Using measurements of Form A and protein concentration, we first determined the initial Moco occupancy of purified VcMCP (22%) as well as EcYiiM (4%), NcNR (50%), and BtXO (50%) (Fig.…”

Section: Moco Bound To Protein Is Stablementioning

confidence: 99%

“…Absorption was measured using a Multiskan GO Microplate Spectrophotometer (Thermo Scientific). Quantification of Moco content of the proteins was conducted using HPLC-based measurements of Form A, a stable and fluorescent Mocooxidation product (Hercher et al 2020a). Stability of protein-bound Moco was assessed by incubating the Moco:protein complexes at various temperatures (4°C, 22°C, and 37°C) for 96 hours.…”

Section: Purification and Characterization Of Moco-binding Proteinsmentioning

confidence: 99%

Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficientC. elegans

Warnhoff

Hercher

Mendel

et al. 2020

Preprint

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The molybdenum cofactor (Moco) is a 520 dalton prosthetic group that is synthesized in a multi-step enzymatic pathway present in most Archaea, Bacteria, and Eukarya. In animals, four oxidases (among them sulfite oxidase) use Moco as a prosthetic group. Moco is essential in animals; humans with mutations in genes that encode Moco-biosynthetic enzymes display lethal neurological and developmental defects. Moco supplementation seems a logical therapy, however the instability of Moco has precluded biochemical and cell biological studies of Moco transport and bioavailability. The nematode Caenorhabditis elegans can take up Moco from its bacterial diet and transport it to cells and tissues that express Moco-requiring enzymes, suggesting a system for Moco uptake and distribution. Here we show that protein-bound Moco is the stable, bioavailable species of Moco taken up by C. elegans from its diet and is an effective dietary supplement in a C. elegans model of Moco deficiency. Diverse purified Moco:protein complexes from bacteria, bread mold, green algae, and dairy cows were able to support the growth of otherwise Moco-deficient C. elegans mutants grown on Moco-deficient E. coli. We show that these Moco:protein complexes are very stable, suggesting they may provide a strategy for the production and delivery of therapeutically active Moco to treat human Moco deficiency.

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“…Oxidation and quantification of cPMP and Moco/MPT was carried out essentially as described previously [16,[34][35][36][37].…”

Section: Quantitative Detection Of Molybdenum Cofactor and Its Metabomentioning

confidence: 99%

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

et al. 2020

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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.

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Insights into the Cnx1E catalyzed MPT-AMP hydrolysis

Cited by 16 publications

References 34 publications

The structure of the Moco carrier protein fromRippkaea orientalis

The structure of the Moco carrier protein fromRippkaea orientalis

Protein-bound molybdenum cofactor is bioavailable and rescues molybdenum cofactor-deficientC. elegans

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

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