title: Structure of a dinuclear iron cluster-containing β-hydroxylase active in antibiotic biosynthesis.
abstract: A family of dinuclear iron cluster-containing oxygenases that catalyze β-hydroxylation tailoring reactions in natural product biosynthesis by nonribosomal peptide synthetase (NRPS) systems was recently described [Makris, T. M., Chakrabarti, M., Münck, E., and Lipscomb, J. D. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 15391-15396]. Here, the 2.17 Å X-ray crystal structure of the archetypal enzyme from the family, CmlA, is reported. CmlA catalyzes β-hydroxylation of l-p-aminophenylalanine during chloramphenicol biosynthesis. The fold of the N-terminal domain of CmlA is unlike any previously reported, but the C-terminal domain has the αββα fold of the metallo-β-lactamase (MBL) superfamily. The diiron cluster bound in the C-terminal domain is coordinated by an acetate, three His residues, two Asp residues, one Glu residue, and a bridging oxo moiety. One of the Asp ligands forms an unusual monodentate bridge. No other oxygen-activating diiron enzyme utilizes this ligation or the MBL protein fold. The N-terminal domain facilitates dimerization, but using computational docking and a sequence-based structural comparison to homologues, we hypothesize that it likely serves additional roles in NRPS recognition and the regulation of O2 activation.
authors: Department of Biochemistry, Molecular Biology and Biophysics and Center for Metals in Biocatalysis, University of Minnesota , Minneapolis, Minnesota 55455, United States.
authors: Thomas M
authors: TM
authors: Makris
authors: Cory J
authors: CJ
authors: Knoot
authors: Carrie M
authors: CM
authors: Wilmot
authors: John D
authors: JD
authors: Lipscomb
mesh_terms: D001665:Binding Sites; D002701:Chloramphenicol / Q000096:biosynthesis; D018360:Crystallography, X-Ray; D006900:Hydroxylation; D007501:Iron / Q000737:chemistry*; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D010453:Peptide Synthases / Q000378:metabolism*
chemical_list: D002701:Chloramphenicol; D007501:Iron; D006899:Mixed Function Oxygenases; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
references: Chem Biol. 1995 Sep;2(9):409-18
references: 9432288
references: J Mol Biol. 1993 Jan 5;229(1):105-24
references: 7678431
references: Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
references: 15299926
references: J Gen Microbiol. 1985 Jan;131(1):97-104
references: 3989509
references: Biochemistry. 2006 Dec 19;45(50):14869-79
references: 17154525
references: Proc Natl Acad Sci U S A. 2008 Oct 14;105(41):15696-701
references: 18838690
references: J Mol Biol. 2010 May 21;398(5):715-29
references: 20359483
references: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references: 15572765
references: Acc Chem Res. 2011 Apr 19;44(4):280-8
references: 21391602
references: Biochemistry. 2008 Aug 12;47(32):8386-97
references: 18627173
references: Nature. 1993 Dec 9;366(6455):537-43
references: 8255292
references: Nat Protoc. 2010 May;5(5):883-97
references: 20431534
references: Science. 2008 Aug 1;321(5889):659-63
references: 18583577
references: Nature. 2005 Feb 10;433(7026):657-61
references: 15654328
references: Nucleic Acids Res. 2010 Jul;38(Web Server issue):W545-9
references: 20457744
references: Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):235-42
references: 21460441
references: J Inorg Biochem. 2006 Apr;100(4):679-93
references: 16504301
references: Science. 2006 Sep 1;313(5791):1291-4
references: 16946071
references: Nat Struct Biol. 2000 Nov;7(11):1041-5
references: 11062560
references: Science. 1995 Jun 9;268(5216):1489-92
references: 7770774
references: Chem Rev. 2006 Aug;106(8):3468-96
references: 16895337
references: Microbiology (Reading). 2001 Oct;147(Pt 10):2817-2829
references: 11577160
references: Chem Rev. 1996 Nov 7;96(7):2625-2658
references: 11848839
references: Methods Enzymol. 1997;276:307-26
references: 27754618
references: Biochemistry. 2002 Jan 8;41(1):42-51
references: 11772001
references: J Mol Evol. 2003 Apr;56(4):435-45
references: 12664163
references: J Biol Chem. 2010 Aug 6;285(32):24562-74
references: 20519494
references: J Biol Chem. 2004 Jul 16;279(29):30600-10
references: 15096510
references: J Biol Chem. 1992 Sep 5;267(25):17588-97
references: 1325441
references: Nucleic Acids Res. 2009 Jan;37(Database issue):D387-92
references: 18931379
references: Acc Chem Res. 2007 Jul;40(7):475-83
references: 17567087
references: J Am Chem Soc. 2001 Feb 7;123(5):827-38
references: 11456616
references: Proc Natl Acad Sci U S A. 2006 Nov 14;103(46):17462-7
references: 17090667
references: Chem Rev. 2003 Jun;103(6):2167-201
references: 12797828
references: Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10037-41
references: 11517324
references: Biochemistry. 2006 Apr 18;45(15):4848-58
references: 16605252
references: Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):213-21
references: 20124702
references: Proc Natl Acad Sci U S A. 2010 Aug 31;107(35):15391-6
references: 20713732
references: J Am Chem Soc. 2004 Mar 31;126(12):3777-88
references: 15038731
references: Biochemistry. 2001 Oct 2;40(39):11651-9
references: 11570865
references: Biochem Biophys Res Commun. 2005 Dec 9;338(1):230-9
references: 16165090
references: Science. 2006 Apr 14;312(5771):273-6
references: 16614225
references: J Am Chem Soc. 2011 May 11;133(18):6938-41
references: 21506543
references: Biochemistry. 2005 May 3;44(17):6492-501
references: 15850383
references: Proc Natl Acad Sci U S A. 2008 Dec 9;105(49):19194-8
references: 19033467
references: Biochem Pharmacol. 2007 Dec 15;74(12):1686-701
references: 17597585
references: Nature. 2013 Feb 21;494(7437):380-4
references: 23395959