Chapter Four - Expression, purification and properties of the enzymes involved in lanthanide-dependent alcohol oxidation: XoxF4, XoxF5, ExaF/PedH, and XoxG4

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Abstract

In this chapter we describe logistics, protocols and conditions for expression, purification and characterization of Ln3 +-dependent alcohol dehydrogenases representing three distinct phylogenetic clades of these enzymes, classified as XoxF4, XoxF5 and ExaF/PedH. We present data on the biochemical properties of a dozen enzymes, all generated by our group, in a comparative fashion. These enzymes display a range of properties in terms of substrate and metal specificities, pH and ammonium requirement, as well as catalytic constants. In addition, we describe a single novel cytochrome, XoxG4, that likely serves as a natural electron acceptor from XoxF5 in methanotrophs of the Gammaproteobacteria class.

Introduction

Lanthanide (Ln3 +)-dependent alcohol dehydrogenases have emerged recently as important and relatively widely distributed enzymes (Chistoserdova, 2016; Chistoserdova & Kalyuzhnaya, 2018; Cotruvo, 2019; Daumann, 2019). While it has been proposed that these enzymes are more catalytically efficient than their calcium (Ca2 +)-dependent counterparts (Keltjens, Pol, Reimann, & Op den Camp, 2014), the question remains whether the two enzymes act independently or in unison. In many organisms, the two alternative forms are present and operating simultaneously, their relative expression subject to multiple regulatory mechanisms (Yu, Zheng, Huang, & Chistoserdova, 2019). As Ln3 +-dependent microbial metabolism is still an emerging area, more data are needed on the properties and the activities of the Ln3 +-dependent enzymes. Notably, Ln3 +-dependent enzymes are extremely diverse, often showing less amino acid conservation with members of a different clade than with their Ca2 +-dependent counterparts (Chistoserdova, 2011; Chistoserdova & Kalyuzhnaya, 2018; Keltjens et al., 2014). Here we describe protocols for expression, purification and analysis of several types of such enzymes. Specifically, we focus on methanol dehydrogenases of two phylogenetic affiliations, XoxF4 and XoxF5, and ethanol dehydrogenase classified as ExaF/PedH. Finally, we describe the properties and the activity of one associated cytochrome, XoxG4.

Section snippets

Gene synthesis and expression vector construction

We originally attempted to express several Ln3 +-dependent alcohol dehydrogenases in Escherichia coli, using commercial pET vectors, but these constructs did not result in production of active enzymes, even when cells were grown in the presence of pyrroloquinoline quinone (PQQ) and Ln3 +, the natural cofactors, or when the expressed proteins were incubated with these cofactors in attempts to reconstitute active holoenzymes (not shown). An alternative approach was to utilize a broad host range

Protein expression in M. extorquens AM1

For proteins expressed in M. extorquens AM1, generally, 300 mL cultures were grown in shake flasks with succinate as a substrate (0.2% w/v) to late exponential phase and collected by centrifugation at 4700 × g for 10 min. Pellets were transferred to 100 mL of fresh minimal medium supplemented with methanol (0.5% v/v) and Ce3 + (30 μM), and these were incubated overnight at 30 °C with shaking at 200 rpm to an OD600 of 0.6–0.8. Cells were harvested by centrifugation at 4700 × g for 15 min at 4 °C, and pellets

pH optimum

To determine optimum pH values, standard buffers were used as follows: 100 mM sodium/potassium phosphate (pH range 6.0–8.0), 100 mM Tris-HCl (pH range 7.5–9.0), 100 mM sodium carbonate/sodium bicarbonate (pH range 9.0–10.0). All the XoxF5 enzymes as well as the single ExaF/PedH enzyme we characterized revealed a pH optimum between 9.0 and 9.5. One of the XoxF4 enzymes revealed a similar pH optimum with a peak at 9.0, while the second revealed a somewhat lower pH optimum, with a peak at 8.5 (Huang

Heterologous expression of alternative cytochromes

As Ln3 +-dependent alcohol dehydrogenases are represented by proteins widely diverging in their sequences (Chistoserdova, 2011; Chistoserdova & Kalyuzhnaya, 2018), it appears that the electron transfer chains also vary widely. At least four divergent cytochromes have been identified among alpha-, beta- and gammaproteobacterial methylotrophs that were highly expressed during growth on methane (Yu, Beck, & Chistoserdova, 2017). In addition, a plastocyanin-encoding gene has been identified as part

Summary

We here presented logistics, protocols and conditions for expression, purification and characterization of Ln3 +-dependent alcohol dehydrogenases from a variety of bacterial sources. We also presented data on the biochemical properties of a dozen enzymes generated by our group, in a comparative fashion, pinpointing some contrasting characteristics. We also briefly described properties of a novel cytochrome that likely serves as a natural electron acceptor from XoxF5 MDH enzymes, acting in

Acknowledgment

This material is based upon work supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research under award DE-SC-0016224.

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