A
            <i>Mycobacterium tuberculosis</i>
            ligand-binding Mn/Fe protein reveals a new cofactor in a remodeled R2-protein scaffold

Andersson, Charlotta S.; Högbom, Martin

doi:10.1073/pnas.0812971106

Cited by 73 publications

(145 citation statements)

References 38 publications

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“…The mixedmetal cofactor activates oxygen and catalyzes the formation of an ether cross-link in the protein scaffold, demonstrating the chemical potential of this cofactor. (Table S1), is very similar to the previously reported structure of its homolog from M. tuberculosis (MtR2lox) (11). The active site architectures of both proteins are virtually identical: The metal ions are coordinated by two histidine and four glutamate residues and bridged by a μ-oxo/hydroxo ligand ( Fig.…”

Section: Significancesupporting

confidence: 71%

“…The protein was found to belong to a novel group of R2-like proteins, denoted R2-like ligand-binding oxidases (R2lox) (11,24). These Significance Metallocofactors enable enzymes to catalyze difficult reactions that would otherwise not be possible, such as the reduction of oxygen.…”

mentioning

confidence: 99%

“…The protein was found to belong to a novel group of R2-like proteins, denoted R2-like ligand-binding oxidases (R2lox) (11,24). These…”

mentioning

confidence: 99%

“…For four decades it was assumed that all ferritin superfamily proteins contained diiron cofactors. However, in recent years new subfamilies harboring either a dimanganese or heterodinuclear Mn/Fe cofactor have been documented (7)(8)(9)(10)(11)(12)(13)(14). The Mn/Fe cofactor was discovered in class Ic RNR R2 subunits, where its Mn IV /Fe III state functionally replaces the diiron-tyrosyl radical cofactor of class Ia R2s (9, 10).…”

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confidence: 99%

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Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein

Griese¹,

Roos

Cox

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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283

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Although metallocofactors are ubiquitous in enzyme catalysis, how metal binding specificity arises remains poorly understood, especially in the case of metals with similar primary ligand preferences such as manganese and iron. The biochemical selection of manganese over iron presents a particularly intricate problem because manganese is generally present in cells at a lower concentration than iron, while also having a lower predicted complex stability according to the Irving-Williams series (Mn II < Fe II < Ni II < Co II < Cu II > Zn II ). Here we show that a heterodinuclear Mn/Fe cofactor with the same primary protein ligands in both metal sites self-assembles from Mn II and Fe II in vitro, thus diverging from the Irving-Williams series without requiring auxiliary factors such as metallochaperones. Crystallographic, spectroscopic, and computational data demonstrate that one of the two metal sites preferentially binds Fe II over Mn II as expected, whereas the other site is nonspecific, binding equal amounts of both metals in the absence of oxygen. Oxygen exposure results in further accumulation of the Mn/Fe cofactor, indicating that cofactor assembly is at least a twostep process governed by both the intrinsic metal specificity of the protein scaffold and additional effects exerted during oxygen binding or activation. We further show that the mixed-metal cofactor catalyzes a two-electron oxidation of the protein scaffold, yielding a tyrosine-valine ether cross-link. Theoretical modeling of the reaction by density functional theory suggests a multistep mechanism including a valyl radical intermediate.H alf of all enzymes are estimated to contain metallocofactors (1). An important subset uses transition metal ions to perform key redox reactions such as oxygen activation. The diiron cofactor of the ferritin-like superfamily of proteins is particularly versatile (2). While ferritin itself simply oxidizes and sequesters iron (3), in other family members the diiron center acts as a transient one-or two-electron oxidant. In the R2 subunits of class I ribonucleotide reductases (RNRs) it generates a redoxactive tyrosyl radical (4, 5), whereas in the bacterial multicomponent monooxygenases (BMMs) it catalyzes the hydroxylation of a variety of hydrocarbons (6). For four decades it was assumed that all ferritin superfamily proteins contained diiron cofactors. However, in recent years new subfamilies harboring either a dimanganese or heterodinuclear Mn/Fe cofactor have been documented (7)(8)(9)(10)(11)(12)(13)(14). The Mn/Fe cofactor was discovered in class Ic RNR R2 subunits, where its Mn IV /Fe III state functionally replaces the diiron-tyrosyl radical cofactor of class Ia R2s (9, 10). After a long controversy, class Ib R2 proteins were shown to use a dimanganese cofactor in the same scaffold (7,8). These recent developments highlight the complexity of correctly identifying the metals that make up native metallocofactors. While the metal preferences of some primary coordination motifs are well known and distinct, others ar...

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Section: Significancesupporting

confidence: 71%

mentioning

confidence: 99%

“…The protein was found to belong to a novel group of R2-like proteins, denoted R2-like ligand-binding oxidases (R2lox) (11,24). These…”

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein

Griese¹,

Roos

Cox

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

283

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“…Glu-28 also forms a hydrogen bond to the bridging solvent molecule. Bridging solvent-derived ligands are commonly found in dinuclear metal sites, including the recently discovered Mn/Fe heterodinuclear site (30). Asp-245 coordinates the two metal ions in a bridging-chelating mode that is not without precedent in other dimetallic enzymes (31,32).…”

Section: Resultsmentioning

confidence: 99%

Mechanism of ADP-ribosylation removal revealed by the structure and ligand complexes of the dimanganese mono-ADP-ribosylhydrolase DraG

Berthold

Wang²,

Nordlund³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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ADP-ribosylation is a ubiquitous regulatory posttranslational modification involved in numerous key processes such as DNA repair, transcription, cell differentiation, apoptosis, and the pathogenic mechanism of certain bacterial toxins. Despite the importance of this reversible process, very little is known about the structure and mechanism of the hydrolases that catalyze removal of the ADP-ribose moiety. In the phototrophic bacterium Rhodospirillum rubrum, dinitrogenase reductase-activating glycohydrolase (DraG), a dimanganese enzyme that reversibly associates with the cell membrane, is a key player in the regulation of nitrogenase activity. DraG has long served as a model protein for ADP-ribosylhydrolases. Here, we present the crystal structure of DraG in the holo and ADP-ribose bound forms. We also present the structure of a reaction intermediate analogue and propose a detailed catalytic mechanism for protein de-ADP-ribosylation involving ring opening of the substrate ribose. In addition, the particular manganese coordination in DraG suggests a rationale for the enzyme's preference for manganese over magnesium, although not requiring a redox active metal for the reaction.binuclear dinuclear ͉ bioinorganic chemistry ͉ covalent modification ͉ nitrogen fixation ͉ posttranslational modifications P osttranslational modification of proteins by the attachment of chemical groups is used by cells from all kingdoms of life and occurs in several forms. Through the reversible covalent modification of specific amino acid side chains, enzyme activity, or protein function can be switched on and off as a rapid response to environmental stimuli. ADP-ribosylation is a posttranslational modification existing in two distinct forms, mono-and poly-ADPribosylation, resulting from the attachment of a single ADP-ribose moiety or a polymeric ADP-ribose chain structure, respectively, to the protein (1, 2).Mono-ADP-ribosylation was first discovered as a process used in the action of several bacterial toxins, including cholera-, diphtheria-, and pertussis toxin (3, 4). The pathogenesis of these diseases involves ADP-ribosylation of specific human proteins, catalyzed by the toxins, thereby affecting the activity of the modified proteins. In human physiology, mono-ADP-ribosylation is found in the regulation of cell-cell and cell-matrix interactions, as well as part of immune function (5, 6). Poly-ADP-ribosylation is involved in many fundamental processes such as DNA repair, transcription and cell differentiation, and is also implicated in carcinogenesis (6-8). Poly-ADP-ribosylation inhibitors are currently in phase II clinical trials for treatment of breast and ovarian cancer (9).Mono-ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs) and is specifically targeted to amino acids residues such as Arg, Asn, Glu, Asp, Cys, diphthamide, or phosphoserine. The reaction is stereospecific, using ␤-NAD ϩ as a substrate, forming the ␣-anomer of the ADP-ribosylated amino acid and releasing nicotinamide (10) (Fig. 1). PolyADP-ribosylation i...

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Comparative structural analysis provides new insights into the function of R2‐like ligand‐binding oxidase

et al. 2022

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R2‐like ligand‐binding oxidase (R2lox) is a ferritin‐like protein that harbours a heterodinuclear manganese–iron active site. Although R2lox function is yet to be established, the enzyme binds a fatty acid ligand coordinating the metal centre and catalyses the formation of a tyrosine–valine ether cross‐link in the protein scaffold upon O2 activation. Here, we characterized the ligands copurified with R2lox by mass spectrometry‐based metabolomics. Moreover, we present the crystal structures of two new homologs of R2lox, from Saccharopolyspora erythraea and Sulfolobus acidocaldarius, at 1.38 Å and 2.26 Å resolution, respectively, providing the highest resolution structure for R2lox, as well as new insights into putative mechanisms regulating the function of the enzyme.

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A Mycobacterium tuberculosis ligand-binding Mn/Fe protein reveals a new cofactor in a remodeled R2-protein scaffold

Cited by 73 publications

References 38 publications

Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein

Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein

Mechanism of ADP-ribosylation removal revealed by the structure and ligand complexes of the dimanganese mono-ADP-ribosylhydrolase DraG

Comparative structural analysis provides new insights into the function of R2‐like ligand‐binding oxidase

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