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dc.contributor.authorLu, Jingnan
dc.contributor.authorBrigham, Christopher J.
dc.contributor.authorPlassmeier, Jens K.
dc.contributor.authorSinskey, Anthony J.
dc.date.accessioned2016-07-22T16:50:11Z
dc.date.available2016-07-22T16:50:11Z
dc.date.issued2014-08
dc.date.submitted2014-05
dc.identifier.issn0175-7598
dc.identifier.issn1432-0614
dc.identifier.urihttp://hdl.handle.net/1721.1/103789
dc.description.abstract2-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 (IC[subscript 50] = 1.2 mM), l-isoleucine (IC[subscript 50] = 2.3 mM), and l-leucine (IC[subscript 50] = 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 (K[subscript M] = 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.en_US
dc.publisherSpringer Berlin Heidelbergen_US
dc.relation.isversionofhttp://dx.doi.org/10.1007/s00253-014-5965-3en_US
dc.rightsCreative Commons Attribution-Noncommercial-Share Alikeen_US
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/4.0/en_US
dc.sourceSpringer Berlin Heidelbergen_US
dc.titleCharacterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16en_US
dc.typeArticleen_US
dc.identifier.citationLu, Jingnan, Christopher J. Brigham, Jens K. Plassmeier, and Anthony J. Sinskey. “Characterization and Modification of Enzymes in the 2-Ketoisovalerate Biosynthesis Pathway of Ralstonia Eutropha H16.” Applied Microbiology and Biotechnology 99, no. 2 (August 1, 2014): 761–774.en_US
dc.contributor.departmentHarvard University--MIT Division of Health Sciences and Technologyen_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Biologyen_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Chemistryen_US
dc.contributor.mitauthorLu, Jingnanen_US
dc.contributor.mitauthorPlassmeier, Jens K.en_US
dc.contributor.mitauthorSinskey, Anthony J.en_US
dc.relation.journalApplied Microbiology and Biotechnologyen_US
dc.eprint.versionAuthor's final manuscripten_US
dc.type.urihttp://purl.org/eprint/type/JournalArticleen_US
eprint.statushttp://purl.org/eprint/status/PeerRevieweden_US
dc.date.updated2016-05-23T12:09:41Z
dc.language.rfc3066en
dc.rights.holderSpringer-Verlag Berlin Heidelberg
dspace.orderedauthorsLu, Jingnan; Brigham, Christopher J.; Plassmeier, Jens K.; Sinskey, Anthony J.en_US
dspace.embargo.termsNen
dc.identifier.orcidhttps://orcid.org/0000-0003-0856-0750
dc.identifier.orcidhttps://orcid.org/0000-0003-1847-440X
dc.identifier.orcidhttps://orcid.org/0000-0002-1015-1270
mit.licenseOPEN_ACCESS_POLICYen_US


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