caa a22 4500 605505128 CHVBK 20210128100620.0 cr unu---uuuuu 210128e20150101xx s 000 0 eng 10.1007/s00253-014-5965-3 doi (NATIONALLICENCE)springer-10.1007/s00253-014-5965-3 Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16 [Elektronische Daten] [Jingnan Lu, Christopher Brigham, Jens Plassmeier, Anthony Sinskey] 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 = 1.2mM), l-isoleucine (IC50 = 2.3mM), and l-leucine (IC50 = 5.4mM). 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 = 10.5μM) 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. Springer-Verlag Berlin Heidelberg, 2014 Ralstonia eutropha nationallicence Branched-chain amino acids nationallicence Acetohydroxy acid synthase nationallicence Acetolactate synthase nationallicence Acetohydroxyacid isomeroreductase nationallicence Dihydroxyacid dehydratase nationallicence Lu Jingnan Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 02139, Cambridge, MA, USA aut Brigham Christopher Department of Bioengineering, University of Massachusetts Dartmouth, 285 Old Westport Road, 02747, North Dartmouth, MA, USA aut Plassmeier Jens Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 02139, Cambridge, MA, USA aut Sinskey Anthony Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 02139, Cambridge, MA, USA aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/2(2015-01-01), 761-774 0175-7598 99:2<761 2015 99 253 https://doi.org/10.1007/s00253-014-5965-3 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-5965-3 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Lu Jingnan Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 02139, Cambridge, MA, USA aut NATIONALLICENCE 700 1- Brigham Christopher Department of Bioengineering, University of Massachusetts Dartmouth, 285 Old Westport Road, 02747, North Dartmouth, MA, USA aut NATIONALLICENCE 700 1- Plassmeier Jens Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 02139, Cambridge, MA, USA aut NATIONALLICENCE 700 1- Sinskey Anthony Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 02139, Cambridge, MA, USA aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/2(2015-01-01), 761-774 0175-7598 99:2<761 2015 99 253