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Switchgrass PviCAD1: Understanding Residues Important for Substrate Preferences and Activity

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Abstract

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in monolignol biosynthesis. Although plants contain numerous genes coding for CADs, only one or two CADs appear to have a primary physiological role in lignin biosynthesis. Much of this distinction appears to reside in a few key residues that permit reasonable catalytic rates on monolignal substrates. Here, several mutant proteins were generated using switchgrass wild type (WT) PviCAD1 as a template to understand the role of some of these key residues, including a proton shuttling HL duo in the active site. Mutated proteins displayed lowered or limited activity on cinnamylaldehydes and exhibited altered kinetic properties compared to the WT enzyme, suggesting that key residues important for efficient catalysis had been identified. We have also shown that a sorghum ortholog containing EW, instead of HL in its active site, displayed negligible activity against monolignals. These results indicate that lignifying CADs require a specific set of key residues for efficient activity against monolignals.

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Acknowledgments

We thank Nathan Palmer for his excellent technical assistance. This work was supported by the USDA-ARS CRIS project 5440-21000-028-00D and in part by the Office of Science (BER), US Department of Energy grant number (DE-AI02-09ER64829). The US Department of Agriculture, Agricultural Research Service, is an equal opportunity/affirmative action employer, and all agency services are available without discrimination. Mention of commercial products and organizations in this manuscript is solely to provide specific information. It does not constitute endorsement by USDA-ARS over other products and organizations not mentioned.

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Authors and Affiliations

  1. USDA-ARS Grain, Forage, and Bioenergy Research Unit, University of Nebraska, 137 Keim Hall, Lincoln, NE, 68583-0937, USA

    Aaron J. Saathoff, Scott Sattler & Gautam Sarath

  2. Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, 4114 Molecular Biology Building, Ames, IA, 50011, USA

    Mark S. Hargrove

  3. Department of Chemistry, Creighton University, 2500 California Plaza, Omaha, NE, 68178, USA

    Eric J. Haas

  4. USDA-ARS Genomics and Gene Discovery Unit, 800 Buchanan St., Albany, CA, 94710, USA

    Christian M. Tobias

  5. Department of Biology, University of Nebraska-Kearney, Kearney, NE, 68849, USA

    Paul Twigg

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  1. Aaron J. Saathoff
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Correspondence to Gautam Sarath.

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Saathoff, A.J., Hargrove, M.S., Haas, E.J. et al. Switchgrass PviCAD1: Understanding Residues Important for Substrate Preferences and Activity. Appl Biochem Biotechnol 168, 1086–1100 (2012). https://doi.org/10.1007/s12010-012-9843-0

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  • DOI: https://doi.org/10.1007/s12010-012-9843-0

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