Mechanistic studies of the cofactor assembly in class Ib ribonucleotide reductases and protein affinity for MnII and FeII

Abstract

Ribonucleotide reductase (RNR) is an essential enzyme found in all organisms. The function of RNR is to catalyze the conversion of nucleotides to deoxynucleotides. RNRs rely on metallocofactors to oxidize a conserved cysteine in the active site of the enzyme into a thiyl radical, which then initiates nucleotide reduction. The proteins required for MnIII2–Y• cluster formation in class Ib RNRs are NrdF (β-subunit) and NrdI (flavodoxin). An oxidant is channeled from the FMN cofactor in NrdI to the dimanganese center in NrdF, where it oxidizes the dimanganese center and a tyrosyl radical (Y•) is formed. Both Streptococcus sanguinis and Escherichia coli MnII2–NrdF structures have a constriction in the channel immediately above the metal site. In E. coli, the constriction is formed by the side chain of S159, whereas in the S. sanguinis system it involves T158. This serine-to-threonine substitution was investigated using S. sanguinis and Streptococcus pneumoniae class Ib RNRs but it is also present in other pathogenic streptococci. Using stopped-flow kinetics, we investigate the role of this substitution in the mechanism of MnIII2–Y• cluster formation. In addition to different kinetics observed in the studied streptococci, we found that affinity constants of NrdF for MnII and FeII are about 1 µM and the previously reported preference for MnII could not be explained by affinity only.

A New Thiolate‐Bound Dimanganese Cluster as a Structural and Functional Model for Class Ib Ribonucleotide Reductases

“…Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides into their corresponding deoxyribonucleotides, which are precursors necessary for DNA synthesis and repair in all organisms. [1][2][3][4][5][6][7] Three different classes of RNRs are known, with class I being subdivided into five subclasses (Ia-e). [1,7,8] The RNRs differ in the nature of the stable metallocofactors required for the transient generation of a cysteine thiyl radical (Cys * ) that initiates nucleotide reduction.…”

“…Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides into their corresponding deoxyribonucleotides, which are precursors necessary for DNA synthesis and repair in all organisms [1–7] . Three different classes of RNRs are known, with class I being subdivided into five subclasses (Ia–e) [1, 7, 8] .…”

Why is manganese so valuable to bacterial pathogens?

“…Surprisingly, a riboregulatory mechanism functionally comparable to the action of RyhB has not been described, so that class Ia RNR expression in E. coli continues in the presence of iron starvation, although this results in a largely inactive population of these enzymes (Cotruvo and Stubbe, 2011). Grave et al (2020) demonstrated that the enzyme intrinsically selects for manganese in both binding sites when metal-free class Ib RNR from B. anthracis is presented with equimolar concentrations of Mn 2+ and Fe 2+ , whereas no intrinsic selectivity was observed in the case of class Ib RNRs from S. sanguinis (Jayachandran et al, 2021). Since the active site consists of two metal binding sites, the question arises to what extent the individual binding sites cooperate to accommodate the cognate metals.…”

“…The idea that cooperativity Distribution of class I RNRs based on their metal-dependency in pathogens. N. gonorrhoeae (Narasimhan et al, 2022), C. jejuni (Alqurashi et al, 2021), S. Typhimurium (Eriksson et al, 1998;Cotruvo and Stubbe, 2010;Panosa et al, 2010;Cotruvo and Stubbe, 2011;Martin and Imlay, 2011), C. trachomatis (Jiang et al, 2007), A. ureae (Rose et al, 2019), S. sanguinis (Makhlynets et al, 2014), S. pneumoniae (Jayachandran et al, 2021), S. pyogenes (Roca et al, 2008), B. cereus (John et al, 2022), B. subtilis (Zhang and Stubbe, 2011), B. anthracis (Grave et al, 2020), M. tuberculosis (Hammerstad et al, 2014). Although the RNR of C. diphtheriae has not been directly characterized, it is very likely that it is manganese-dependent, as two close non-pathogenic relatives, C. glutamicum and C. ammoniagenes, have been demonstrated to be manganese-dependent RNRs (Abbouni et al, 2009;Cox et al, 2010).…”

Ein neuer Thiolat‐gebundener Dimangan‐Cluster als strukturelles und funktionales Modell der Class Ib Ribonukleotidreduktasen

“…Ribonukleotidreduktasen (RNRs) katalysieren die Umwandlung von Ribonukleotiden zu ihren entsprechenden Desoxyribonukleotiden, die wichtige Vorstufen für die DNA‐Synthese und ‐Reparatur in allen Organsimen sind [1–7] . Drei verschiedene Klassen der RNRs sind bekannt, wobei die Class I in fünf Unterklassen unterteilt wird (I a–e) [1, 7, 8] .…”