Characterization of a Rhodobacter sphaeroides primary fatty acid kinase
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Characterization of a Rhodobacter sphaeroides primary fatty acid kinase Maxwell J. Bachochin1 · Michelle Van Allen1 · Robert D. Barber1 Received: 1 August 2020 / Revised: 3 September 2020 / Accepted: 15 September 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Widely distributed among prokaryotes, short chain fatty acid kinases provide a path for fatty acid entry into central metabolic pathways. These enzymes catalyze the reversible, ATP-dependent synthesis of acyl-phosphates, which leads to the production of acyl-CoA derivatives by a coordinate acyltransferase. To date, characterized representatives of short chain fatty acid kinases exhibit relatively narrow substrate specificity. In this work, biochemical characterization of a predicted acetate kinase from Rhodobacter sphaeroides reveals a novel enzyme with broad substrate specificity for primary fatty acids of varying lengths (C2-–C8). Keywords Rhodobacter sphaeroides · Acetate kinase · Polyhydroxybutyrate · ASKHA
Introduction Short chain fatty acid kinases are typically classified based upon amino acid sequence similarity to enzymes that demonstrate activity with specific substrates such as acetate, propionate, or butyrate. The R. sphaeroides genome sequence is predicted to contain a single gene (ackA) encoding a short chain fatty acid kinase (RsAck; KEGG ID: RSP_1254) that shares substantial sequence identity to characterized acetate kinases (Kontur et al. 2012; Aceti and Ferry 1988; Matsuyama et al. 1989; Chittori et al. 2013). To test this prediction, ackA was cloned from R. sphaeroides str. 2.4.1 genomic DNA into an expression vector and the recombinant hexahistidine tagged RsAck variant was heterologously produced in Escherichia coli. The recombinant enzyme was purified to homogeneity using nickel affinity chromatography and substrate specificity was assessed.
Communicated by Johann Heider. * Robert D. Barber [email protected] 1
Department of Biological Sciences, College of Natural and Health Sciences, University of Wisconsin-Parkside, 900 Wood Rd, Kenosha, WI 53141‑2000, USA
Heterologous expression and purification of RsAck Initially, a DNA fragment encoding ackA was PCR-amplified from R. sphaeroides 2.4.1 genomic DNA (NCBI Accession: NC_007493.2) using 2X PCR Master Mix (Promega) and cloned directly into the expression vector pQE30UA using the forward DNA primer 5′-ATGAAACCCGTCTGGCTC GTCCTGAAC-3′ and reverse DNA primer 5′-CGAAAG ACCGGTCAGGCAAGCGC-3′. The resulting PCR-amplified, gel-purified DNA fragment was ligated into pQE30UA vector (50 ng/µl, QIAGEN) using 2X Ligation Master Mix (QIAGEN). Later, in an effort to improve protein production, an EcoRV and HindIII (New England Biolabs) DNA fragment was excised from the pQE30UA construct and ligated into a EcoRV and HindIII-digested pET30b using Clonables and Novablue competent cells (Novagen). Restriction enzyme analysis and DNA sequencing (Arizona State University Core Facilities, Tempe, AZ) were performed to confirm clones at ea
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