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

Wajmann, Simon; Hercher, Thomas W.; Buchmeier, Sabine; Hänsch, Robert; Mendel, Ralf R.; Kruse, Tobias

doi:10.3390/microorganisms8040534

Cited by 6 publications

(3 citation statements)

References 54 publications

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“…The N. crassa wild-type strain and the NCU01356 strain were cultivated according to [37]. Next, 10 6 conidia was used to inoculate a 2 L flask and was subsequently incubated for 20 h at 30 • C and 140 rpm.…”

Section: Neurospora Crassa Preparationmentioning

confidence: 99%

Precise Quantification of Molybdate In Vitro by the FRET-Based Nanosensor ‘MolyProbe’

et al. 2022

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Molybdenum (Mo) is an essential trace element in all kingdoms of life. Mo is bioavailable as the oxyanion molybdate and gains biological activity in eukaryotes when bound to molybdopterin, forming the molybdenum cofactor. The imbalance of molybdate homeostasis results in growth deficiencies or toxic symptoms within plants, fungi and animals. Recently, fluorescence resonance energy transfer (FRET) methods have emerged, monitoring cellular and subcellular molybdate distribution dynamics using a genetically encoded molybdate-specific FRET nanosensor, named MolyProbe. Here, we show that the MolyProbe system is a fast and reliable in vitro assay for quantitative molybdate determination. We added a Strep-TagII affinity tag to the MolyProbe protein for quick and easy purification. This MolyProbe is highly stable, resistant to freezing and can be stored for several weeks at 4 °C. Furthermore, the molybdate sensitivity of the assay peaked at low nM levels. Additionally, The MolyProbe was applied in vitro for quantitative molybdate determination in cell extracts of the plant Arabidopsis thaliana, the fungus Neurospora crassa and the yeast Saccharomyces cerevisiae. Our results show the functionality of the Arabidopsis thaliana molybdate transporter MOT1.1 and indicate that FRET-based molybdate detection is an excellent tool for measuring bioavailable Mo.

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Section: Neurospora Crassa Preparationmentioning

confidence: 99%

Precise Quantification of Molybdate In Vitro by the FRET-Based Nanosensor ‘MolyProbe’

et al. 2022

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“…Recent work identified 3′,8‐cH 2 GTP as initial, short‐life, first step intermediate (Hover et al, 2013 , 2015 ). Subsequent work identified its formation to be under tight transcriptional control—at least in the fungus Neurospora crassa (Wajmann et al, 2020 ), suggesting that regulation of Moco biosynthesis is realized at the earliest stage possible. In the second pathway step cPMP is converted into molybdopterin (MPT), a tricyclic pterin derivative (Mendel & Kruse, 2012 ; Wuebbens & Rajagopalan, 2003 ).…”

Section: Introductionmentioning

confidence: 99%

The structural principles underlying molybdenum insertase complex assembly

Hassan,

Ihling,

Iacobucci

et al. 2023

Protein Science

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Within the cell, the trace element molybdenum (Mo) is only biologically active when complexed either within the nitrogenase specific FeMo cofactor or within the molybdenum cofactor (Moco). Moco consists of an organic part, called molybdopterin (MPT) and an inorganic part, i.e. the Mo‐center. The enzyme which catalyses the Mo‐center formation is the molybdenum insertase (Mo‐insertase). Mo‐insertases consist of two functional domains called G‐ and E‐domain. The G‐domain catalyses the formation of adenylated MPT (MPT‐AMP), which is the substrate for the E‐domain, that catalyses the actual molybdate insertion reaction. Though the functions of E‐ and G‐domain have been elucidated to great structural and mechanistic detail, their combined function is poorly characterized. In this work, we describe a structural model of the eukaryotic Mo‐insertase Cnx1 complex that was generated based on cross‐linking mass spectrometry combined with computational modelling. We revealed Cnx1 to form an asymmetric hexameric complex which allows the E‐ and G‐domain active sites to align in a catalytic productive orientation towards each other.This article is protected by copyright. All rights reserved.

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“…Within the first step of the pathway, GTP is converted to cyclic pyranopterin monophosphate (cPMP, [ 1 , 2 ]) a reaction thatinvolves the formation of the instable intermediate 3′,8-cH 2 GTP [ 3 , 4 ]. Recent work suggests that it is the first step of eukaryotic Moco biosynthesis which is under regulatory control as deduced from the finding that in the filamentous fungus Neurospora crassa ( N. crassa , [ 5 ]) the 3′,8-cH 2 GTP synthesizing enzyme is co-regulated (precisely the formation of one particular splice variant) with the major N. crassa Moco user enzyme (i.e. nitrate reductase).…”

Section: Introductionmentioning

confidence: 99%

Moco Carrier and Binding Proteins

Kruse

2022

Molecules

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The molybdenum cofactor (Moco) is the active site prosthetic group found in numerous vitally important enzymes (Mo-enzymes), which predominantly catalyze 2 electron transfer reactions. Moco is synthesized by an evolutionary old and highly conserved multi-step pathway, whereby the metal insertion reaction is the ultimate reaction step here. Moco and its intermediates are highly sensitive towards oxidative damage and considering this, they are believed to be permanently protein bound during synthesis and also after Moco maturation. In plants, a cellular Moco transfer and storage system was identified, which comprises proteins that are capable of Moco binding and release but do not possess a Moco-dependent enzymatic activity. The first protein described that exhibited these properties was the Moco carrier protein (MCP) from the green alga Chlamydomonas reinhardtii. However, MCPs and similar proteins have meanwhile been described in various plant species. This review will summarize the current knowledge of the cellular Moco distribution system.

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

Cited by 6 publications

References 54 publications

Precise Quantification of Molybdate In Vitro by the FRET-Based Nanosensor ‘MolyProbe’

Precise Quantification of Molybdate In Vitro by the FRET-Based Nanosensor ‘MolyProbe’

The structural principles underlying molybdenum insertase complex assembly

Moco Carrier and Binding Proteins

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