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Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16

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Abstract

2-Ketoisovalerate is an important cellular intermediate for the synthesis of branched-chain amino acids as well as other important molecules, such as pantothenate, coenzyme A, and glucosinolate. This ketoacid can also serve as a precursor molecule for the production of biofuels, pharmaceutical agents, and flavor agents in engineered organisms, such as the betaproteobacterium Ralstonia eutropha. The biosynthesis of 2-ketoisovalerate from pyruvate is carried out by three enzymes: acetohydroxyacid synthase (AHAS, encoded by ilvBH), acetohydroxyacid isomeroreductase (AHAIR, encoded by ilvC), and dihydroxyacid dehydratase (DHAD, encoded by ilvD). In this study, enzymatic activities and kinetic parameters were determined for each of the three R. eutropha enzymes as heterologously purified proteins. AHAS, which serves as a gatekeeper for the biosynthesis of all three branched-chain amino acids, demonstrated the tightest regulation through feedback inhibition by l-valine (IC50 = 1.2 mM), l-isoleucine (IC50 = 2.3 mM), and l-leucine (IC50 = 5.4 mM). Intermediates in the valine biosynthesis pathway also exhibit feedback inhibitory control of the AHAS enzyme. In addition, AHAS has a very weak affinity for pyruvate (KM = 10.5 μM) and is highly selective towards 2-ketobutyrate (R = 140) as a second substrate. AHAIR and DHAD are also inhibited by the branched-chain amino acids, although to a lesser extent when compared to AHAS. Experimental evolution and rational site-directed mutagenesis revealed mutants of the regulatory subunit of AHAS (IlvH) (N11S, T34I, A36V, T104S, N11F, G14E, and N29H), which, when reconstituted with wild-type IlvB, lead to AHAS having reduced valine, leucine, and isoleucine sensitivity. The study of the kinetics and inhibition mechanisms of R. eutropha AHAS, AHAIR, and DHAD has shed light on interactions between these enzymes and the products they produce; it, therefore, can be used to engineer R. eutropha strains with optimal production of 2-ketoisovalerate for value-added materials.

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Acknowledgments

The authors thank Dr. Claudia Santos Gai for helpful discussions, Mr. John W. Quimby for critical review of this manuscript, and Mr. Caio Alves for assistance with R. eutropha experimental evolution experiment. This work is funded by the U.S. Department of Energy, Advanced Research Projects Agency-Energy (ARPA-E). JL is supported by the Malaysian Palm Oil Board.

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Authors and Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139, USA

    Jingnan Lu

  2. Department of Bioengineering, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, MA, 02747, USA

    Christopher J. Brigham

  3. Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139, USA

    Jens K. Plassmeier & Anthony J. Sinskey

  4. Division of Health Sciences and Technology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139, USA

    Anthony J. Sinskey

  5. Engineering Systems Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139, USA

    Anthony J. Sinskey

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  1. Jingnan Lu
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  4. Anthony J. Sinskey
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Correspondence to Anthony J. Sinskey.

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Lu, J., Brigham, C.J., Plassmeier, J.K. et al. Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16. Appl Microbiol Biotechnol 99, 761–774 (2015). https://doi.org/10.1007/s00253-014-5965-3

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