Altering the Substrate Specificity of Acetyl-CoA Synthetase by Rational Mutagenesis of the Carboxylate Binding Pocket

dc.contributor.author Nikolau, Basil
dc.contributor.author Yandeau-Nelson, Marna
dc.contributor.author Sofeo, Naazneen
dc.contributor.author Hart, Jason
dc.contributor.author Butler, Brandon
dc.contributor.author Oliver, David
dc.contributor.author Oliver, David
dc.contributor.author Yandeau-Nelson, Marna
dc.contributor.author Nikolau, Basil
dc.contributor.department NSF Engineering Research Center for Biorenewable Chemicals
dc.contributor.department Genetics, Development and Cell Biology
dc.contributor.department Center for Metabolic Biology
dc.contributor.department NSF Engineering Research Center for Biorenewable Chemicals
dc.date 2019-09-14T12:50:40.000
dc.date.accessioned 2020-06-30T04:02:01Z
dc.date.available 2020-06-30T04:02:01Z
dc.date.copyright Tue Jan 01 00:00:00 UTC 2019
dc.date.issued 2019-01-01
dc.description.abstract <p>Acetyl-CoA synthetase (ACS) is a member of a large superfamily of enzymes that display diverse substrate specificities, with a common mechanism of catalyzing the formation of a thioester bond between Coenzyme A and a carboxylic acid, while hydrolyzing ATP to AMP and pyrophosphate. As an activated form of acetate, acetyl-CoA is a key metabolic intermediate that links many metabolic processes, including the TCA cycle, amino acid metabolism, fatty acid metabolism and biosynthetic processes that generate many polyketides and some terpenes. We explored the structural basis of the specificity of ACS for only activating acetate, whereas other members of this superfamily utilize a broad range of other carboxylate substrates. By computationally modeling the structure of the <em>Arabidopsis</em> ACS and the <em>Pseudomonas chlororaphis</em> isobutyryl-CoA synthetase using the experimentally determined tertiary structures of homologous ACS enzymes as templates, we identified residues that potentially comprise the carboxylate binding pocket. These predictions were systematically tested by mutagenesis of four specific residues. The resulting rationally redesigned carboxylate binding pocket modified the size and chemo-physical properties of the carboxylate binding pocket. This redesign successfully switched a highly specific enzyme from using only acetate, to be equally specific for using longer linear (up to hexanoate) or branched chain (methylvalerate) carboxylate substrates. The significance of this achievement is that it sets a precedent for understanding the structure–function relationship of an enzyme without the need for an experimentally determined tertiary structure of that target enzyme, and rationally generates new biocatalysts for metabolic engineering of a broad range of metabolic processes.</p>
dc.description.comments <p>This article is published as Sofeo, Naazneen, Jason H. Hart, Brandon Butler, David J. Oliver, Marna D. Yandeau-Nelson, and Basil J. Nikolau. "Altering the substrate specificity of acetyl-CoA synthetase by rational mutagenesis of the carboxylate binding pocket." <em>ACS synthetic biology</em> (2019). doi: <a href="https://doi.org/10.1021/acssynbio.9b00008" title="DOI URL">10.1021/acssynbio.9b00008</a>.</p>
dc.format.mimetype application/pdf
dc.identifier archive/lib.dr.iastate.edu/gdcb_las_pubs/224/
dc.identifier.articleid 1228
dc.identifier.contextkey 14665593
dc.identifier.s3bucket isulib-bepress-aws-west
dc.identifier.submissionpath gdcb_las_pubs/224
dc.identifier.uri https://dr.lib.iastate.edu/handle/20.500.12876/37899
dc.language.iso en
dc.source.bitstream archive/lib.dr.iastate.edu/gdcb_las_pubs/224/2019_Nelson_AlteringSubstrate.pdf|||Fri Jan 14 22:43:17 UTC 2022
dc.source.uri 10.1021/acssynbio.9b00008
dc.subject.disciplines Biochemistry, Biophysics, and Structural Biology
dc.subject.disciplines Cell and Developmental Biology
dc.subject.disciplines Computational Biology
dc.subject.disciplines Genetics and Genomics
dc.subject.keywords acetyl-CoA synthetase
dc.subject.keywords targeted mutagenesis
dc.subject.keywords enzyme engineering
dc.subject.keywords substrate specificity
dc.subject.keywords homologous enzymes
dc.subject.keywords Arabidopsis
dc.title Altering the Substrate Specificity of Acetyl-CoA Synthetase by Rational Mutagenesis of the Carboxylate Binding Pocket
dc.type article
dc.type.genre article
dspace.entity.type Publication
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