title: Mechanistic insights from the binding of substrate and carbocation intermediate analogues to aristolochene synthase.
abstract: Aristolochene synthase, a metal-dependent sesquiterpene cyclase from Aspergillus terreus, catalyzes the ionization-dependent cyclization of farnesyl diphosphate (FPP) to form the bicyclic eremophilane (+)-aristolochene with perfect structural and stereochemical precision. Here, we report the X-ray crystal structure of aristolochene synthase complexed with three Mg(2+) ions and the unreactive substrate analogue farnesyl-S-thiolodiphosphate (FSPP), showing that the substrate diphosphate group is anchored by metal coordination and hydrogen bond interactions identical to those previously observed in the complex with three Mg(2+) ions and inorganic pyrophosphate (PPi). Moreover, the binding conformation of FSPP directly mimics that expected for productively bound FPP, with the exception of the precise alignment of the C-S bond with regard to the C10-C11 π system that would be required for C1-C10 bond formation in the first step of catalysis. We also report crystal structures of aristolochene synthase complexed with Mg(2+)3-PPi and ammonium or iminium analogues of bicyclic carbocation intermediates proposed for the natural cyclization cascade. Various binding orientations are observed for these bicyclic analogues, and these orientations appear to be driven by favorable electrostatic interactions between the positively charged ammonium group of the analogue and the negatively charged PPi anion. Surprisingly, the active site is sufficiently flexible to accommodate analogues with partially or completely incorrect stereochemistry. Although this permissiveness in binding is unanticipated, based on the stereochemical precision of catalysis that leads exclusively to the (+)-aristolochene stereoisomer, it suggests the ability of the active site to enable controlled reorientation of intermediates during the cyclization cascade. Taken together, these structures illuminate important aspects of the catalytic mechanism.
authors.affiliation: Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, USA.
authors.forename: Mengbin
authors.initials: M
authors.lastname: Chen
authors.forename: Naeemah
authors.initials: N
authors.lastname: Al-lami
authors.forename: Marine
authors.initials: M
authors.lastname: Janvier
authors.forename: Edward L
authors.initials: EL
authors.lastname: D'Antonio
authors.forename: Juan A
authors.initials: JA
authors.lastname: Faraldos
authors.forename: David E
authors.initials: DE
authors.lastname: Cane
authors.forename: Rudolf K
authors.initials: RK
authors.lastname: Allemann
authors.forename: David W
authors.initials: DW
authors.lastname: Christianson
mesh_terms: D001230:Aspergillus / Q000201:enzymology / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002413:Cations, Divalent; D011756:Diphosphates / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D007535:Isomerases / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D011106:Polyisoprenyl Phosphates / Q000737:chemistry / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism
chemical_list: D002413:Cations, Divalent; D011756:Diphosphates; D011106:Polyisoprenyl Phosphates; D000644:Quaternary Ammonium Compounds; D012717:Sesquiterpenes; C054277:aristolochene; C004808:farnesyl pyrophosphate; D007535:Isomerases; C060050:aristolochene synthase; D008274:Magnesium
references.citation: J Biol Chem. 2008 May 30;283(22):15431-9
references.pmid: 18385128
references.citation: Biochemistry. 2009 Jul 7;48(26):6175-83
references.pmid: 19489610
references.citation: Proc Natl Acad Sci U S A. 2001 Nov 20;98(24):13543-8
references.pmid: 11698643
references.citation: J Am Chem Soc. 2011 Sep 7;133(35):13906-9
references.pmid: 21815676
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Jan;66(Pt 1):12-21
references.pmid: 20057044
references.citation: J Biol Chem. 2004 Mar 5;279(10):8526-9
references.pmid: 14672944
references.citation: Science. 1996 Jan 12;271(5246):163-8
references.pmid: 8539615
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references.pmid: 15572765
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):213-21
references.pmid: 20124702
references.citation: Org Biomol Chem. 2008 Jul 7;6(13):2346-54
references.pmid: 18563268
references.citation: Nat Prod Rep. 2012 Jan;29(1):60-71
references.pmid: 22068697
references.citation: J Org Chem. 2003 Jun 27;68(13):5403-6
references.pmid: 12816509
references.citation: Science. 2012 May 25;336(6084):1030-3
references.pmid: 22628654
references.citation: Pure Appl Chem. 2010;82(8):1585-1597
references.pmid: 21562622
references.citation: Curr Opin Chem Biol. 2008 Apr;12(2):141-50
references.pmid: 18249199
references.citation: Proc Natl Acad Sci U S A. 2002 Nov 26;99(24):15375-80
references.pmid: 12432096
references.citation: Org Lett. 2011 Mar 4;13(5):1202-5
references.pmid: 21271717
references.citation: J Biol Chem. 1993 Feb 25;268(6):4543-8
references.pmid: 8440737
references.citation: Methods Enzymol. 1997;276:307-26
references.pmid: 27754618
references.citation: Chem Rev. 2006 Aug;106(8):3412-42
references.pmid: 16895335
references.citation: Biochemistry. 2003 Jul 1;42(25):7741-7
references.pmid: 12820883
references.citation: Structure. 1998 Feb 15;6(2):127-33
references.pmid: 9519404
references.citation: Chem Commun (Camb). 2002 Oct 21;(20):2384-5
references.pmid: 12430452
references.citation: J Am Chem Soc. 2004 Jun 16;126(23):7212-21
references.pmid: 15186158
references.citation: Biochemistry. 2010 Mar 2;49(8):1787-97
references.pmid: 20131801
references.citation: J Org Chem. 2010 Feb 19;75(4):1119-25
references.pmid: 20095558
references.citation: Nat Chem Biol. 2011 May 22;7(7):431-3
references.pmid: 21602811
references.citation: J Org Chem. 2009 Apr 3;74(7):2631-45
references.pmid: 19323569
references.citation: Science. 1997 Sep 19;277(5333):1815-20
references.pmid: 9295271
references.citation: Science. 1997 Sep 19;277(5333):1820-4
references.pmid: 9295272
references.citation: J Am Chem Soc. 2002 Oct 2;124(39):11636-41
references.pmid: 12296728
references.citation: J Biol Chem. 2000 Aug 18;275(33):25533-9
references.pmid: 10825154
references.citation: Science. 1997 Sep 19;277(5333):1811-5
references.pmid: 9295270
references.citation: Biochemistry. 2007 Feb 20;46(7):1941-51
references.pmid: 17261032
references.citation: Chem Commun (Camb). 2012 Mar 28;48(26):3230-2
references.pmid: 22349314
references.citation: Phytochemistry. 2009 Oct-Nov;70(15-16):1621-37
references.pmid: 19793600
references.citation: J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
references.pmid: 19461840
references.citation: Biochemistry. 2005 Apr 26;44(16):6153-63
references.pmid: 15835903
references.citation: J Org Chem. 2000 Nov 17;65(23):8093-5
references.pmid: 11073627
references.citation: Acc Chem Res. 2013 Apr 16;46(4):885-93
references.pmid: 23214924