Semiquinone-induced Maturation of Bacillus anthracis Ribonucleotide Reductase by a Superoxide Intermediate

Berggren, Gustav; Duraffourg, Nicolas; Sahlin, Margareta; Sjöberg, Britt‐Marie

doi:10.1074/jbc.m114.592535

Cited by 16 publications

(25 citation statements)

References 32 publications

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“…It is important to note that the reduced Mn II /Mn II cofactor of class Ib and Id R2 proteins does not directly react with O2 (9,13,19). Instead, superoxide is thought to be the in vivo oxidant (14,19,44). In the G68L-R2lox system, Fenton-like chemistry of free Fe II in solution could instead be the source of superoxide.…”

Section: G68l-r2lox Predominantly Assembles An R2clike Mn III /Fe Iiimentioning

confidence: 99%

Chemical flexibility of heterobimetallic Mn/Fe cofactors: R2lox and R2c proteins

Kutin

Kositzki

Branca

et al. 2019

Journal of Biological Chemistry

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Section: G68l-r2lox Predominantly Assembles An R2clike Mn III /Fe Iiimentioning

confidence: 99%

Chemical flexibility of heterobimetallic Mn/Fe cofactors: R2lox and R2c proteins

Kutin

Kositzki

Branca

et al. 2019

Journal of Biological Chemistry

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“…The tyrosyl radical then initiates ribonucleotide reduction in the R1 subunit [ 6 , 8 , 9 ]. In class Ib RNRs, a dimanganese cofactor undergoes similar reactions, although it is activated by superoxide, which is provided by an additional flavodoxin subunit [ 11 – 17 ]. In the recently proposed class Id RNRs, which also bind a dimanganese cofactor, the radical equivalent appears to be stored at the metal site rather than at a tyrosine [ 18 , 19 ].…”

Section: Introductionmentioning

confidence: 99%

Assembly of a heterodinuclear Mn/Fe cofactor is coupled to tyrosine–valine ether cross-link formation in the R2-like ligand-binding oxidase

et al. 2019

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R2-like ligand-binding oxidases (R2lox) assemble a heterodinuclear Mn/Fe cofactor which performs reductive dioxygen (O 2 ) activation, catalyzes formation of a tyrosine–valine ether cross-link in the protein scaffold, and binds a fatty acid in a putative substrate channel. We have previously shown that the N-terminal metal binding site 1 is unspecific for manganese or iron in the absence of O 2 , but prefers manganese in the presence of O 2 , whereas the C-terminal site 2 is specific for iron. Here, we analyze the effects of amino acid exchanges in the cofactor environment on cofactor assembly and metalation specificity using X-ray crystallography, X-ray absorption spectroscopy, and metal quantification. We find that exchange of either the cross-linking tyrosine or the valine, regardless of whether the mutation still allows cross-link formation or not, results in unspecific manganese or iron binding at site 1 both in the absence or presence of O 2 , while site 2 still prefers iron as in the wild-type. In contrast, a mutation that blocks binding of the fatty acid does not affect the metal specificity of either site under anoxic or aerobic conditions, and cross-link formation is still observed. All variants assemble a dinuclear trivalent metal cofactor in the aerobic resting state, independently of cross-link formation. These findings imply that the cross-link residues are required to achieve the preference for manganese in site 1 in the presence of O 2 . The metalation specificity, therefore, appears to be established during the redox reactions leading to cross-link formation. Electronic supplementary material The online version of this article (10.1007/s00775-019-01639-4) contains supplementary material, which is available to authorized users.

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“…To calculate the total yield of protein, Beer‐Lamberts Law is used. The extinction coefficient for the oxidized, protein‐bound FMN cofactor in NrdI at 450 nm ( ε 450ox ) is 10.8 mM −1 cm −1 . The resulting protein yield varies for different semesters and student groups, averaging at 2–10 mg per L culture medium.…”

Section: Resultsmentioning

confidence: 99%

A Research‐inspired biochemistry laboratory module–combining expression, purification, crystallization, structure‐solving, and characterization of a flavodoxin‐like protein

Hammerstad

Røhr

Hersleth

2019

Biochem Molecular Bio Educ

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Many laboratory courses consist of short and seemingly unconnected individual laboratory exercises. To increase the course consistency, relevance, and student engagement, we have developed a research‐inspired and project‐based module, “From Gene to Structure and Function”. This 2.5‐week full‐day biochemistry and structural biology module covers protein expression, purification, structure solving, and characterization. The module is centered around the flavodoxin‐like protein NrdI, involved in the activation of the bacterial ribonucleotide reductase enzyme system. Through an in‐depth focus on one specific protein, the students will learn the basic laboratory skills needed in order to generate a broader knowledge and breadth within the field. With respect to generic skills, the students report their findings as a scientific article, with the aim to learn to present concise research results and write scientific papers. The current research‐inspired project has the potential of being further developed into a more discovery‐driven project and extended to include other molecular biological techniques or biochemical/biophysical characterizations. In student evaluations, this research‐inspired laboratory course has received very high ratings and been highly appreciated, where the students have gained research experience for more independent future work in the laboratory. © 2019 The Authors. Biochemistry and Molecular Biology Education published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 47(3):318–332, 2019.

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Semiquinone-induced Maturation of Bacillus anthracis Ribonucleotide Reductase by a Superoxide Intermediate

Cited by 16 publications

References 32 publications

Chemical flexibility of heterobimetallic Mn/Fe cofactors: R2lox and R2c proteins

Chemical flexibility of heterobimetallic Mn/Fe cofactors: R2lox and R2c proteins

Assembly of a heterodinuclear Mn/Fe cofactor is coupled to tyrosine–valine ether cross-link formation in the R2-like ligand-binding oxidase

A Research‐inspired biochemistry laboratory module–combining expression, purification, crystallization, structure‐solving, and characterization of a flavodoxin‐like protein

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