Chapter One - Preparation of hydroxycinnamoyl-coenzyme A thioesters using recombinant 4-coumarate:coenzyme A ligase (4CL) for characterization of BAHD hydroxycinnamoyltransferase enzyme activities

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Abstract

Analyses of the enzymatic activities of hydroxycinnamoyl-coenzyme A (CoA) hydroxycinnamoyltransferases of the BAHD family require hydroxycinnamoyl-CoA thioesters as assay reagents. Here we describe a simple, cost-effective method for preparing p-coumaroyl-, caffeoyl- and feruloyl-CoA thioesters using the Arabidopsis thaliana 4-coumarate:CoA ligase 1 (4CL1) expressed in Escherichia coli. Preparation of the 4CL enzyme, in vitro synthesis of the thioesters, and thioester purification utilizing a C-18 solid phase extraction column are detailed. The hydroxycinnamoyl-CoA thioesters produced are suitable for downstream qualitative and quantitative analyses.

Introduction

The BAHD acyltransferases are a large enzyme family that use acyl-coenzyme A (CoA) thioester donors as substrates (D'auria, 2006). The name is derived from the first letter of the names of the first four enzymes of this family to be biochemically characterized (benzylalcohol O-acetyltransfearse [BEAT], anthocyanin O-hydroxycinnamoyltransferase [AHCT], anthraniliate N-hydroxycinnamoyl/benzoyltransferase [HCBT], and deacetylvindoline 4-O-acetyltransferase [DAT]). One clade of the BAHD family, Vb, is comprised of hydroxycinnamoyl-CoA hydroxycinnamoyltransferases (D'auria, 2006; Tuominen, Johnson, & Tsai, 2011), which transfer the hydroxycinnamoyl moiety from a CoA donor to either hydroxyl or amine groups on appropriate acceptors to form the corresponding ester or amide (Fig. 1, Reaction II). Members of this clade have diverse roles in plant growth and development, including biosynthesis of intermediates in phenylpropanoid and monolignol biosynthetic pathways (Hoffmann et al., 2004; Shadle et al., 2007), modification of cell wall components (Marita, Hatfield, Rancour, & Frost, 2014), and accumulation of specialized metabolites that may help protect plants against biotic and abiotic stress (Niggeweg, Michael, & Martin, 2004; Sullivan & Knollenberg, 2021; Wise, 2013). Although many Clade Vb BAHD hydroxycinnamoyl-CoA hydroxycinnamoyltransferases have been identified by bioinformatics, many, if not most, of these have yet to be characterized biochemically.

Characterization of the enzymatic activities of hydroxycinnamoyl-CoA hydroxycinnamoyltransferases in vitro requires appropriate hydroxycinnamoyl-CoA donor substrates. Early methods to produce these utilized partially purified enzyme preparations capable of carrying out the CoA ligase reaction (Gross & Zenk, 1966; Lindl, Kreuzaler, & Hahlbrock, 1973), but producing sufficient amounts of hydroxycinnamoyl-CoA substrates for some applications was challenging. Both Johns (1974), and Stoeckigt and Zenk (1975) developed chemical syntheses for these, with the latter also determining more accurate spectrophotometric parameters for them, in order to produce the larger quantities needed for hydroxycinnamoyl-CoA hydroxycinnamoyltransferase characterization. Unfortunately, some researchers may find chemical synthesis of hydroxycinnamoyl-CoA substrates daunting, especially if they have little or no background in synthetic organic chemistry. One alternative is to purchase hydroxycinnamoyl-CoA thioester substrates. Depending on the quantity needed for the research, however, this can be costly. At this writing, based on data from the American Chemical Society's SciFinder website (https://scifinder.cas.org), prices ranged from $50–130 per μmol for p-coumaroyl-CoA and $70–110 per μmol for caffeoyl-CoA. Improvements in enzymatic synthesis have been made, with one described by Zenk, Ulbrich, Busse, and Stoeckigt (1979) that used an enzyme preparation from Pseudomonas putida, and another from Hatfield et al. (2009) that employed 4-coumarate CoA ligase (4CL) activity in maize rind extracts (Fig. 1, Reaction I). The latter, developed by Dr. Jane Marita, may have been based in part on the method described by Zenk et al. (1979), but utilized a simple and effective C-18 solid-phase extraction (SPE) column for purification of the reaction products. The ability to express large amounts of active enzymes in Escherichia. coli, however, eliminates much work associated with using native 4CL enzyme sources with a supply of 4CL enzyme to last several years produced in a few hours of hands-on time.

The procedure described below derives from the method described by Hatfield et al. (2009), except it utilizes recombinant Arabidopsis thaliana 4CL1 protein and subsequent modifications to the C-18 SPE procedure by both Dr. Marita and us. We routinely use the procedure to prepare p-coumaroyl-, caffeoyl-, and feruloyl-CoA thioesters of sufficient quantity and purity for qualitative and quantitative (for example, measurement of kinetic parameters [Sullivan & Bonawitz, 2018]) analysis of BAHD hydroxycinnamoyl-CoA hydroxycinnamoyltransferases, as well as for use in BAHD transferase-mediated synthesis of mg quantities of hydroxycinnamoyl esters or amides of interest (see for example, Sullivan & Knollenberg, 2021). The procedure is simple, and at this writing, costs under $5 per μmol in materials to produce.

Section snippets

Expression of arabidopsis 4CL1 in E. coli

4-Coumarate coenzyme A ligase (4CL) is prepared by expression in E. coli. Although 4CL is encoded by a multigene family in A. thaliana, 4CL1 (Lee, Ellard, Wanner, Davis, & Douglas, 1995) seems to best utilize multiple hydroxycinnamic acids. It can accept trans p-coumaric, caffeic, and ferulic acids particularly well as substrates, and to a lesser degree cinnamic, and 5-hydroxyferulic acids (Costa et al., 2005). Sinapic acid does not appear to be a substrate for 4CL1. Our expression construct

Summary

The procedure outlined above, utilizing recombinant arabidopsis 4CL1 protein to synthesize hydroxycinnamoyl-CoA thioesters in vitro, followed by a simple C-18 SPE purification, provides a simple, low-cost approach to preparing this reagent for use in biochemical characterization of BAHD hydroxycinnamoyl-CoA transferases. Quality of the prepared reagent is appropriate for both qualitative and quantitative (e.g., determination of kinetic parameters as described in (cite other chapter)) studies.

Acknowledgments

This chapter is dedicated to the memory of Jane Marita, who contributed substantially to development of the methods and protocols described here. I wish to thank Laurie Reinhardt for assistance with LC analysis of hydroxycinnamoyl-CoA products and for, along with Amanda Fanelli and Levi Svaren, helpful comments on the manuscript. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or

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    2023, Methods in Enzymology
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    Prepare fresh daily Hydroxycinnamoyl-CoA (synthesized chemically, purchased, or prepared enzymatically as described elsewhere in this volume (Sullivan, 2023)) Acceptor substrate (here, shikimic or quinic acid)

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