YcfDRM is a thermophilic oxygen-dependent ribosomal protein uL16 oxygenase

Rok Sekirnik,Christopher J Schofield,Hanna Tarhonskaya,Martin Münzel,Aayan Hussein,Michael A Mcdonough,Christoph Loenarz,Sarah E Wilkins,Emily Flashman,Shabaz Mohammed,Jacob Bush

doi:10.1007/s00792-018-1016-9

Abstract

YcfD from Escherichia coli is a homologue of the human ribosomal oxygenases NO66 and MINA53, which catalyse histidyl-hydroxylation of the 60S subunit and affect cellular proliferation (Ge et al., Nat Chem Biol 12:960–962, 2012). Bioinformatic analysis identified a potential homologue of ycfD in the thermophilic bacterium Rhodothermus marinus (ycfDRM). We describe studies on the characterization of ycfDRM, which is a functional 2OG oxygenase catalysing (2S,3R)-hydroxylation of the ribosomal protein uL16 at R82, and which is active at significantly higher temperatures than previously reported for any other 2OG oxygenase. Recombinant ycfDRM manifests high thermostability (Tm 84 °C) and activity at higher temperatures (Topt 55 °C) than ycfDEC (Tm 50.6 °C, Topt 40 °C). Mass spectrometric studies on purified R. marinus ribosomal proteins demonstrate a temperature-dependent variation in uL16 hydroxylation. Kinetic studies of oxygen dependence suggest that dioxygen availability can be a limiting factor for ycfDRM catalysis at high temperatures, consistent with incomplete uL16 hydroxylation observed in R. marinus cells. Overall, the results that extend the known range of ribosomal hydroxylation, reveal the potential for ycfD-catalysed hydroxylation to be regulated by temperature/dioxygen availability, and that thermophilic 2OG oxygenases are of interest from a biocatalytic perspective.

Highlights

Ferrous iron and 2-oxoglutarate (2OG)-dependent oxygenases (2OG oxygenases) are a ubiquitous enzyme superfamily that catalyses a wide range of oxidative reactions (Hausinger and Schofield 2015)
Intrigued by the similarity of factor inhibiting HIF (FIH) to apparent bacterial JmjC proteins, we recently identified Escherichia coli ycfD (ycfDEC) as an arginyl hydroxylase, which catalyses hydroxylation of R81 in the E. coli 50S ribosomal protein uL16 (Ge et al 2012) [ribosomal nomenclature is as according to Ban et al (2014)]
Inset: deconvoluted ESI–MS spectrum showing partial hydroxylation of ribosomal protein u L16RM. c MS/MS spectrum of u L16RM fragment showing hydroxylation at R82; d Hydroxylation of u L16RM decreases with an increase in growth temperature of R. marinus and varies for growth in baffled (2.5 L polypropylene filled to 1 L with media) and unbaffled flasks (2 L Pyrex filled to 0.6 L with media). uL16RM intact protein masses were determined by ESI–MS spectrometry

Summary

Introduction

Ferrous iron and 2-oxoglutarate (2OG)-dependent oxygenases (2OG oxygenases) are a ubiquitous enzyme superfamily that catalyses a wide range of oxidative reactions (Hausinger and Schofield 2015). Their biochemical roles are diverse and include the catalysis of steps in secondary metabolite biosynthesis in plants and microbes and the regulation of transcription in most, if not all, eukaryotes. In humans and other animals, 2OG oxygenases play a central role in coordinating the cellular and physiological responses to limiting oxygen levels (hypoxia) (Hausinger and Schofield 2015; Schofield and Ratcliffe 2004). The HIF pathway appears to be limited to animals (Loenarz et al 2011); in microbes, other well-characterised mechanisms not involving 2OG oxygenases are established as hypoxia sensors (Taabazuing et al 2014)

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Conclusion