Eukaryotic Molybdenum Insertases

Kruse, Tobias

doi:10.1002/9781119951438.eibc2736

Cited by 5 publications

(9 citation statements)

References 40 publications

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“…This leads to the conclusion that the Mo first coordination sphere geometry present in Moco-AMP is also fully compatible with all cellular downstream processes that Moco is subjected to, including Moco transfer, storage and insertion into apo-enzymes 47 - 49 . Given the high degree of structural and functional conservation amongst eukaryotic Mo-insertases 21 , 28 , we expect that the reaction mechanism described in this work is valid for all eukaryotic Mo-insertases.…”

Section: Discussionmentioning

confidence: 90%

“…Work on the first and second steps of the Moco biosynthesis pathway were primarily performed using E. coli enzymes, which serve as model enzymes here 20 . Elucidation of the third and fourth steps in the pathway primarily involved the A. thaliana molybdenum insertase (Mo-insertase) Cnx1 serving as a model enzyme 21 . Early work with fungal Mo-insertase mutants identified these to be functionally rescued when grown in the presence of millimolar concentrations of sodium molybdate 22 - 24 , rendering molybdate complexation by the MPT dithiolene to be independent from an enzyme catalyst under conditions where excess molybdate is available.…”

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confidence: 99%

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Mechanism of molybdate insertion into pterin-based molybdenum cofactors

et al. 2021

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The molybdenum cofactor (Moco) is found in the active site of numerous important enzymes that are critical to biological processes. The bidentate ligand that chelates molybdenum (Mo) in Moco is the pyranopterin dithiolene (molybdopterin, MPT); however, neither the mechanism of molybdate insertion into MPT nor the structure of Moco prior to its insertion into pyranopterin molybdenum enzymes is known. Here we report this final maturation step, where adenylated MPT (MPT-AMP) and molybdate are the substrates. X-ray crystallography of the Arabidopsis thaliana Mo-insertase variant Cnx1E S269D D274S identified adenylated Moco (Moco-AMP) as an unexpected intermediate in this reaction sequence. X-ray absorption spectroscopy revealed the first coordination sphere geometry of Moco trapped in the Cnx1E active site. We have used this structural information to deduce a mechanism for molybdate insertion into MPT-AMP. Given their high degree of structural and sequence similarity, we suggest that this mechanism is employed by all eukaryotic Mo-insertases.

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Section: Discussionmentioning

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Mechanism of molybdate insertion into pterin-based molybdenum cofactors

et al. 2021

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“…The term Mo-insertases commonly describes enzymes which possess molybdotransferase activity (EC 2.10.1.1) and/or molybdopterin adenylyltransferase activity (EC 2.7.7.75) [ 34 ]. However, the EC nomenclature is rarely used in the literature.…”

Section: Catalyzed Reactions Of Molybdenum Insertasesmentioning

confidence: 99%

Function of Molybdenum Insertases

Kruse

2022

Molecules

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For most organisms molybdenum is essential for life as it is found in the active site of various vitally important molybdenum dependent enzymes (Mo-enzymes). Here, molybdenum is bound to a pterin derivative called molybdopterin (MPT), thus forming the molybdenum cofactor (Moco). Synthesis of Moco involves the consecutive action of numerous enzymatic reaction steps, whereby molybdenum insertases (Mo-insertases) catalyze the final maturation step, i.e., the metal insertion reaction yielding Moco. This final maturation step is subdivided into two partial reactions, each catalyzed by a distinctive Mo-insertase domain. Initially, MPT is adenylylated by the Mo-insertase G-domain, yielding MPT-AMP which is used as substrate by the E-domain. This domain catalyzes the insertion of molybdate into the MPT dithiolene moiety, leading to the formation of Moco-AMP. Finally, the Moco-AMP phosphoanhydride bond is cleaved by the E-domain to liberate Moco from its synthesizing enzyme. Thus formed, Moco is physiologically active and may be incorporated into the different Mo-enzymes or bind to carrier proteins instead.

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“…As such, MPT serves as a substrate for the molybdenum‐insertase (Mo‐insertase, recently reviewed in Kruse, 2022a ) which catalyzes the third and fourth step of Moco biosynthesis. Mo‐insertases are each composed of a functional G‐ and E‐domain (Kruse, 2020 ; Mendel & Kruse, 2012 ). In prokaryotes, these occur as separate entities (Leimkühler et al, 2011 ), while—with the known exception of the green alga Chlamydomonas reinhardtii (Llamas et al, 2007 )—they are fused in eukaryotes.…”

Section: Introductionmentioning

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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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Eukaryotic Molybdenum Insertases

Cited by 5 publications

References 40 publications

Mechanism of molybdate insertion into pterin-based molybdenum cofactors

Mechanism of molybdate insertion into pterin-based molybdenum cofactors

Function of Molybdenum Insertases

The structural principles underlying molybdenum insertase complex assembly

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