title: The role of distant mutations and allosteric regulation on LovD active site dynamics.
abstract: Natural enzymes have evolved to perform their cellular functions under complex selective pressures, which often require their catalytic activities to be regulated by other proteins. We contrasted a natural enzyme, LovD, which acts on a protein-bound (LovF) acyl substrate, with a laboratory-generated variant that was transformed by directed evolution to accept instead a small free acyl thioester and no longer requires the acyl carrier protein. The resulting 29-mutant variant is 1,000-fold more efficient in the synthesis of the drug simvastatin than the wild-type LovD. This is to our knowledge the first nonpatent report of the enzyme currently used for the manufacture of simvastatin as well as the intermediate evolved variants. Crystal structures and microsecond-scale molecular dynamics simulations revealed the mechanism by which the laboratory-generated mutations free LovD from dependence on protein-protein interactions. Mutations markedly altered conformational dynamics of the catalytic residues, obviating the need for allosteric modulation by the acyl carrier LovF.
authors.affiliation: 1] Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, California, USA. [2].
authors.forename: Gonzalo
authors.identifier: 0000000301054337
authors.initials: G
authors.lastname: Jiménez-Osés
authors.affiliation: 1] Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, California, USA. [2].
authors.forename: Sílvia
authors.initials: S
authors.lastname: Osuna
authors.affiliation: 1] Department of Chemical and Biomolecular Engineering, University of California-Los Angeles, Los Angeles, California, USA. [2].
authors.forename: Xue
authors.initials: X
authors.lastname: Gao
authors.affiliation: Molecular Biology Institute, University of California-Los Angeles, Los Angeles, California, USA.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Sawaya
authors.affiliation: Codexis, Inc., Redwood City, California, USA.
authors.forename: Lynne
authors.initials: L
authors.lastname: Gilson
authors.affiliation: Codexis, Inc., Redwood City, California, USA.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Collier
authors.affiliation: Codexis, Inc., Redwood City, California, USA.
authors.forename: Gjalt W
authors.initials: GW
authors.lastname: Huisman
authors.affiliation: Molecular Biology Institute, University of California-Los Angeles, Los Angeles, California, USA.
authors.forename: Todd O
authors.initials: TO
authors.lastname: Yeates
authors.affiliation: 1] Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, California, USA. [2] Department of Chemical and Biomolecular Engineering, University of California-Los Angeles, Los Angeles, California, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Tang
authors.affiliation: Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, California, USA.
authors.forename: K N
authors.initials: KN
authors.lastname: Houk
mesh_terms: D000494:Allosteric Regulation; D001230:Aspergillus / Q000201:enzymology*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics*; D008148:Lovastatin / Q000096:biosynthesis; D056004:Molecular Dynamics Simulation; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics*; D009154:Mutation; D011487:Protein Conformation
chemical_list: D005656:Fungal Proteins; D009097:Multienzyme Complexes; D008148:Lovastatin
references.citation: Curr Opin Chem Biol. 1999 Feb;3(1):54-9
references.pmid: 10021399
references.citation: Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
references.pmid: 15299926
references.citation: Science. 1999 May 21;284(5418):1368-72
references.pmid: 10334994
references.citation: Nucleic Acids Res. 2008 Jul 1;36(Web Server issue):W233-8
references.pmid: 18442991
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501
references.pmid: 20383002
references.citation: Methods Enzymol. 2003;374:300-21
references.pmid: 14696379
references.citation: Nat Biotechnol. 2007 Mar;25(3):338-44
references.pmid: 17322872
references.citation: Science. 2013 Jan 18;339(6117):307-10
references.pmid: 23258409
references.citation: PLoS One. 2011;6(5):e19230
references.pmid: 21603656
references.citation: J Comput Chem. 2004 Jul 15;25(9):1157-74
references.pmid: 15116359
references.citation: Appl Environ Microbiol. 2007 Apr;73(7):2054-60
references.pmid: 17277201
references.citation: Proc Natl Acad Sci U S A. 2011 Aug 23;108(34):14115-20
references.pmid: 21831831
references.citation: J Mol Graph. 1996 Feb;14(1):33-8, 27-8
references.pmid: 8744570
references.citation: Chem Biol. 2009 Oct 30;16(10):1064-74
references.pmid: 19875080
references.citation: J Mol Biol. 2006 Jul 21;360(4):893-906
references.pmid: 16784753
references.citation: J Chem Theory Comput. 2013 Apr 9;9(4):2126-2136
references.pmid: 23585741
references.citation: J Am Chem Soc. 2009 Jun 24;131(24):8388-9
references.pmid: 19530726
references.citation: Science. 2010 Jul 16;329(5989):305-9
references.pmid: 20558668
references.citation: Proteins. 2001 Sep 1;44(4):400-17
references.pmid: 11484218
references.citation: Angew Chem Int Ed Engl. 2013 May 27;52(22):5700-25
references.pmid: 23526810
references.citation: Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W96-9
references.pmid: 15215358
references.citation: Nature. 1992 Mar 5;356(6364):83-5
references.pmid: 1538787
references.citation: Proteins. 2003 Sep 1;52(4):609-23
references.pmid: 12910460
references.citation: Proc Natl Acad Sci U S A. 2013 Apr 30;110(18):7270-5
references.pmid: 23576739
references.citation: J Chem Phys. 2005 Feb 1;122(5):54101
references.pmid: 15740304
references.citation: Science. 2006 Mar 3;311(5765):1258-62
references.pmid: 16513975
references.citation: Science. 2007 Apr 13;316(5822):254-61
references.pmid: 17431175
references.citation: Science. 2010 Oct 15;330(6002):341-6
references.pmid: 20947758
references.citation: Science. 2011 Oct 28;334(6055):517-20
references.pmid: 22034434
references.citation: Proteins. 2006 Nov 1;65(2):392-406
references.pmid: 16933295
references.citation: Proteins. 2010 Nov 15;78(15):3124-30
references.pmid: 20818657
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2210-21
references.pmid: 15572774
references.citation: Proc Natl Acad Sci U S A. 2005 May 10;102(19):6807-12
references.pmid: 15811945
references.citation: Science. 2003 Aug 29;301(5637):1196-202
references.pmid: 12947189
references.citation: Science. 2011 Apr 8;332(6026):234-8
references.pmid: 21474759
references.citation: Biochemistry. 2010 Mar 16;49(10):2186-93
references.pmid: 20136099
references.citation: J Chem Phys. 2010 Mar 21;132(11):114110
references.pmid: 20331284
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):125-32
references.pmid: 20124692
references.citation: Bioinformatics. 2004 Jan 1;20(1):45-50
references.pmid: 14693807
references.citation: Proc Natl Acad Sci U S A. 2012 Oct 23;109(43):17406-11
references.pmid: 23045647
references.citation: J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
references.pmid: 19461840
references.citation: Protein Sci. 1993 Sep;2(9):1511-9
references.pmid: 8401235
references.citation: Protein Sci. 2002 Mar;11(3):467-78
references.pmid: 11847270