title: Comprehensive Structural and Biochemical Analysis of the Terminal Myxalamid Reductase Domain for the Engineered Production of Primary Alcohols.
abstract: The terminal reductase (R) domain from the non-ribosomal peptide synthetase (NRPS) module MxaA in Stigmatella aurantiaca Sga15 catalyzes a non-processive four-electron reduction to produce the myxalamide family of secondary metabolites. Despite widespread use in nature, a lack of structural and mechanistic information concerning reductive release from polyketide synthase (PKS) and NRPS assembly lines principally limits our ability to redesign R domains with altered or improved activity. Here we report crystal structures for MxaA R, both in the absence and, for the first time, in the presence of the NADPH cofactor. Molecular dynamics simulations were employed to provide a deeper understanding of this domain and further identify residues critical for structural integrity, substrate binding, and catalysis. Aggregate computational and structural findings provided a basis for mechanistic investigations and, in the process, delivered a rationally altered variant with improved activity toward highly reduced substrates.
authors.affiliation: Department of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine, Irvine, CA 92697, USA.
authors.forename: Jesus F
authors.initials: JF
authors.lastname: Barajas
authors.affiliation: Joint Bioenergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA; QB3 Institute, University of California, Berkeley, Berkeley, CA 94270, USA.
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Phelan
authors.affiliation: Department of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine, Irvine, CA 92697, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Schaub
authors.affiliation: Department of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine, Irvine, CA 92697, USA.
authors.forename: Jaclyn T
authors.initials: JT
authors.lastname: Kliewer
authors.affiliation: Department of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine, Irvine, CA 92697, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Kelly
authors.affiliation: Department of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine, Irvine, CA 92697, USA.
authors.forename: David R
authors.initials: DR
authors.lastname: Jackson
authors.affiliation: Department of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine, Irvine, CA 92697, USA.
authors.forename: Ray
authors.initials: R
authors.lastname: Luo
authors.affiliation: Joint Bioenergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA; QB3 Institute, University of California, Berkeley, Berkeley, CA 94270, USA; Department of Chemical and Biomolecular Engineering and Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. Electronic address: keasling@berkeley.edu.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: Department of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine, Irvine, CA 92697, USA. Electronic address: sctsai@uci.edu.
authors.forename: Shiou-Chuan
authors.initials: SC
authors.lastname: Tsai
mesh_terms: D000438:Alcohols / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D056004:Molecular Dynamics Simulation; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism; D010453:Peptide Synthases / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D011090:Polyenes / Q000737:chemistry; D048630:Polyketide Synthases / Q000737:chemistry* / Q000378:metabolism*; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D020606:Stigmatella aurantiaca / Q000201:enzymology / Q000378:metabolism
chemical_list: D000438:Alcohols; D011090:Polyenes; D009249:NADP; D048630:Polyketide Synthases; D010088:Oxidoreductases; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase