This is a repository copy of The flavodoxin FldA activates the class Ia ribonucleotide reductase of Campylobacter jejuni. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/173101/ Version: Published Version Article: Alqurashi, A., Alfs, L., Swann, J. et al. (2 more authors) (2021) The flavodoxin FldA activates the class Ia ribonucleotide reductase of Campylobacter jejuni. Molecular Microbiology. ISSN 0950-382X https://doi.org/10.1111/mmi.14715 Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/ Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ Received: 29 October 2020 | Revised: 11 March 2021 DOI: 10.1111/mmi.14715 RESEARCH ARTICLE The flavodoxin FldA activates the class Ia ribonucleotide reductase of Campylobacter jejuni Abdulmajeed Alqurashi1 | Laura Alfs2 | Jordan Swann2 | Julea N. Butt2 | David J. Kelly 1 1Department of Molecular Biology and Biotechnology, The University of Sheffield, Abstract Sheffield, UK Campylobacter jejuni is a microaerophilic zoonotic pathogen with an atypical res- 2 School of Chemistry, University of East piratory Complex I that oxidizes a flavodoxin (FldA) instead of NADH. FldA is es- Anglia, Norwich, UK sential for viability and is reduced via pyruvate and 2- oxoglutarate oxidoreductases Correspondence (POR/OOR). Here, we show that FldA can also be reduced by FqrB (Cj0559), an David J. Kelly, Department of Molecular Biology and Biotechnology, The University NADPH:FldA reductase. An fqrB deletion mutant was viable but displayed a signifi- of Sheffield, Firth Court, Western Bank, cant growth defect. FqrB is related to flavoprotein reductases from Gram- positive Sheffield S10 2TN, UK. Email: [email protected] bacteria that can reduce NrdI, a specialized flavodoxin that is needed for tyrosyl radi- cal formation in NrdF, the beta subunit of class 1b-type (Mn ) ribonucleotide reduc- Funding information Ministry of Education - Kingdom of Saudi tase (RNR). However, C. jejuni possesses a single class Ia- type (Fe) RNR (NrdAB) that Arabi; UK Biotechnology and Biological would be expected to be ferredoxin dependent. We show that CjFldA is an unusually Sciences Research Council, Grant/Award Number: BB/S002499/1 high potential flavodoxin unrelated to NrdI, yet growth of the fqrB mutant, but not the wild- type or a complemented strain, was stimulated by low deoxyribonucleoside (dRNS) concentrations, suggesting FldA links FqrB and RNR activity. Using purified proteins, we confirmed the NrdB tyrosyl radical could be regenerated in an NADPH, FqrB, and FldA dependent manner, as evidenced by both optical and electron para- magnetic resonance (EPR) spectroscopy. Thus, FldA activates RNR in C. jejuni, partly explaining its essentiality. KEYWORDS Cj0559, flavin, flavodoxin reductase, FqrB, tyrosyl radical 1 | INTRODUCTION sources) with both host/source- specific and generalist strains capa- ble of rapid transition between the different physical, nutritional, Campylobacteriosis is the most common food- borne bacterial zoo- and immunological niches of each host or environment. In humans, nosis worldwide, resulting in a huge public health and economic C. jejuni is an intestinal mucosal pathogen that causes an acute burden, with Campylobacter jejuni, as the major causative agent. bloody diarrhea, severe abdominal cramping, and fever, with ~0.1% Undercooked chicken is the main source of human campylobacte- of infections leading to transient paralysis (and occasional deaths) riosis in many countries; 60%– 80% of cases may be attributable to due to inflammatory neuropathies (e.g., Guillain– Barré syndrome). poulty reservoirs (Sheppard et al., 2009). However, campylobacters In addition, antimicrobial resistance is a major problem, with an are also found in other farm animals and the environment (e.g., water increasing prevalence of fluoroquinolone resistance in particular This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd. Molecular Microbiology. 2021;00:1–16. wileyonlinelibrary.com/journal/mmi | 1 2 | ALQURASHI ET AL. (Haldenby et al., 2020), and C. jejuni has been designated a World A study in the closely related Helicobacter pylori (St Maurice Health Organization (WHO) priority organism for developing novel et al., 2007) reported that a flavin adenine dinucleotide (FAD) con- antimicrobial agents. New insights into C. jejuni biology are needed if taining and NADPH oxidizing enzyme named FqrB (HP1164) could control measures are to be put in place that more effectively reduce also act as a flavodoxin reductase in addition to POR. However, chicken colonization and/or food- chain contamination. based on in vitro studies, these authors proposed that the physi- C. jejuni is a microaerophile, adapted for growth at low oxygen ological function of FqrB was in the production of NADPH via the niches in the host, with most strains unable to grow at atmospheric reversed reaction, that is, the transfer of electrons from pyruvate oxygen levels. Compared with conventional aerobes, C. jejuni is to NADPH via POR, flavodoxin and FrqB. In H. pylori, FqrB is seem- unique in utilizing oxidant- labile enzymes in central metabolic path- ingly an essential enzyme as attempts to produce an fqrB deletion ways, which are critical for growth (Kendall et al., 2014). In particular, mutant were unsuccessful (St Maurice et al., 2007). A homologue of it employs pyruvate and 2- oxoglutarate: acceptor oxidoreductases FqrB (Cj0559) is also present in C. jejuni. Here, we show that CjFldA (POR and OOR), rather than oxygen- stable NAD- linked 2- oxoacid is an unusually high- potential flavodoxin, making NADP reduction dehydrogenases, to transfer substrate- derived electrons to the re- unfavorable. Instead, we demonstrate that FqrB provides an addi- spiratory chain (Taylor & Kelly, 2019; Weerakoon & Olson, 2008). tional route for flavodoxin reduction using NADPH as the ultimate These enzymes contain Fe– S clusters vulnerable to oxidative dam- electron donor and that both FqrB and FldA are involved in tyrosine age; we have previously shown that exposure of C. jejuni cells to radical formation in, and thus activation of, the C. jejuni RNR. prolonged aeration causes their inactivation in vivo, which may be a major contributor to the microaerophilic phenotype (Kendall et al., 2014). Conversely, we could not demonstrate consistent 2 | RESULTS growth under strictly anaerobic conditions, even in the presence of alternative electron acceptors that do allow growth under oxygen- 2.1 | Redox properties of the C. jejuni flavodoxin: limiting conditions (Sellars et al., 2002). This seems to be due to the CjFldA is an unusually high redox potential flavodoxin presence of a single class Ia- type ribonucleotide reductase (RNR) that can be reduced by both POR and OOR that would require catalytic amounts of oxygen to sustain DNA synthesis. The C. jejuni FldA was overproduced in Escherichia coli as a C- The electron acceptor for OOR has been shown to be the fla- terminally his- tagged protein and purified by nickel- affinity and vodoxin FldA (Cj1382; CjFldA), which has a flavin mononucleotide hydrophobic interaction chromatography (HIC) (Figure S1a). The as- (FMN) cofactor. FldA is the sole flavodoxin encoded in the -ge purified (oxidized) protein has a typical flavodoxin absorption spec- nome of C. jejuni NCTC 11168 (Parkhill et al., 2000; Weerakoon & trum (Figure 1a; raw data for all figures in Table S1) with maxima Olson, 2008). Reduced FldA is thought to be reoxidized by donation at ~380 and ~460 nm that matched those of a previous study report- of electrons from FMN to the membrane bound Complex I of the ing the purification of this protein (Weerakoon & Olson, 2008). The respiratory chain. In the canonical Complex I found in well stud- semireduced protein displays a shift of the 380 peak to 360 nm and ied “model” bacteria, the nuoE and nuoF genes encode an NADH- broad absorbance maxima at ~460 and ~600 nm, which are charac- binding module that delivers electrons to Fe– S centers in the NuoG teristics of neutral semiquinone (Figure 1a). The latter was exploited subunit. However, in C. jejuni, these genes are replaced by two novel in optically monitored potentiometric titrations (Figure 1b), to de- genes cj1575c (nuoX) and cj1574c (nuoY), the function of which fine the flavodoxin reduction potentials at pH 7 of −170 mV susver are unknown (Calderon- Gomez et al., 2017; Smith et al., 2000). standard hydrogen electrode (SHE) for the oxidized- semiquinone Conceivably, they may form a docking site for reduced FldA to de- transition (Q/SQ or E2) and −190 mV versus SHE for the semiquinone- liver electrons to the Fe– S clusters of NuoG (Taylor & Kelly, 2019; hydroquinone transition (SQ/HQ or E1). Weerakoon & Olson, 2008) but may also have roles independent of Independent assessment of the redox properties of