title: Characterizing the promiscuity of LigAB, a lignin catabolite degrading extradiol dioxygenase from Sphingomonas paucimobilis SYK-6.
abstract: LigAB from Sphingomonas paucimobilis SYK-6 is the only structurally characterized dioxygenase of the largely uncharacterized superfamily of Type II extradiol dioxygenases (EDO). This enzyme catalyzes the oxidative ring-opening of protocatechuate (3,4-dihydroxybenzoic acid or PCA) in a pathway allowing the degradation of lignin derived aromatic compounds (LDACs). LigAB has also been shown to utilize two other LDACs from the same metabolic pathway as substrates, gallate, and 3-O-methyl gallate; however, kcat/KM had not been reported for any of these compounds. In order to assess the catalytic efficiency and get insights into the observed promiscuity of this enzyme, steady-state kinetic analyses were performed for LigAB with these and a library of related compounds. The dioxygenation of PCA by LigAB was highly efficient, with a kcat of 51 s(-1) and a kcat/KM of 4.26 × 10(6) M(-1)s(-1). LigAB demonstrated the ability to use a variety of catecholic molecules as substrates beyond the previously identified gallate and 3-O-methyl gallate, including 3,4-dihydroxybenzamide, homoprotocatechuate, catechol, and 3,4-dihydroxybenzonitrile. Interestingly, 3,4-dihydroxybenzamide (DHBAm) behaves in a manner similar to that of the preferred benzoic acid substrates, with a kcat/Km value only ∼4-fold lower than that for gallate and ∼10-fold higher than that for 3-O-methyl gallate. All of these most active substrates demonstrate mechanistic inactivation of LigAB. Additionally, DHBAm exhibits potent product inhibition that leads to an inactive enzyme, being more highly deactivating at lower substrate concentration, a phenomena that, to our knowledge, has not been reported for another dioxygenase substrate/product pair. These results provide valuable catalytic insight into the reactions catalyzed by LigAB and make it the first Type II EDO that is fully characterized both structurally and kinetically.
authors: Department of Chemistry, Wesleyan University , 52 Lawn Avenue, Middletown, Connecticut 06459, United States.
authors: Kevin P
authors: KP
authors: Barry
authors: Erika A
authors: EA
authors: Taylor
mesh_terms: D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000009:adverse effects / Q000302:isolation & purification / Q000378:metabolism*; D049308:Dioxygenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D062385:Hydroxybenzoates / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008031:Lignin / Q000378:metabolism*; D010105:Oxygenases / Q000378:metabolism*; D020578:Sphingomonas / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D062385:Hydroxybenzoates; C009091:protocatechuic acid; D008031:Lignin; D007501:Iron; D010105:Oxygenases; D049308:Dioxygenases; C439587:extradiol dioxygenase
references: J Biol Chem. 2002 Jan 18;277(3):2019-27
references: 11707443
references: J Bacteriol. 1990 May;172(5):2704-9
references: 2185230
references: Plant J. 2005 Dec;44(5):718-29
references: 16297065
references: J Bacteriol. 1999 Jan;181(2):531-40
references: 9882667
references: J Biochem. 2004 Jun;135(6):721-30
references: 15213248
references: J Bacteriol. 1993 Jul;175(14):4414-26
references: 8392511
references: J Bacteriol. 1996 Oct;178(20):5930-7
references: 8830689
references: J Am Chem Soc. 2003 Oct 1;125(39):11780-1
references: 14505375
references: FEBS Lett. 1989 Jun 5;249(2):348-52
references: 2737293
references: J Biol Chem. 2005 Oct 21;280(42):35382-90
references: 16030014
references: Science. 2007 Apr 20;316(5823):453-7
references: 17446402
references: J Biol Chem. 2009 Apr 10;284(15):9937-46
references: 19234303
references: Nature. 1960 Nov 12;188:560-6
references: 13719300
references: Nature. 1962 Mar 3;193:842-4
references: 13875598
references: Biochem J. 1968 Oct;109(4):559-68
references: 5683506
references: Arch Microbiol. 1999 Nov;172(5):330-9
references: 10550475
references: J Bacteriol. 1997 Aug;179(15):4841-9
references: 9244273
references: Structure. 1999 Aug 15;7(8):953-65
references: 10467151
references: Biosci Biotechnol Biochem. 2004 Jul;68(7):1434-41
references: 15277747
references: J Biol Inorg Chem. 2011 Feb;16(2):341-55
references: 21153851
references: Biosci Biotechnol Biochem. 2007 Jan;71(1):1-15
references: 17213657
references: Biochemistry. 2005 May 3;44(17):6383-91
references: 15850372
references: Biochemistry. 2009 Jul 14;48(27):6469-81
references: 19518059
references: Nucleic Acids Res. 1990 Jun 11;18(11):3405-6
references: 2356133
references: J Bacteriol. 1996 Sep;178(17):5249-56
references: 8752345
references: Microbiology (Reading). 1998 Apr;144 ( Pt 4):955-963
references: 9579069
references: Biochemistry. 2011 Nov 29;50(47):10262-74
references: 22011290
references: J Biochem. 1978 Apr;83(4):1125-34
references: 26671
references: Biochemistry. 2001 May 29;40(21):6379-87
references: 11371200
references: J Bacteriol. 1996 Nov;178(21):6227-32
references: 8892823
references: J Biol Chem. 2002 Apr 5;277(14):11866-72
references: 11805095
references: Appl Environ Microbiol. 1981 May;41(5):1159-65
references: 7259155
references: Biochemistry. 2011 Oct 18;50(41):8899-913
references: 21870805
references: FEBS Lett. 1990 Nov 26;275(1-2):53-7
references: 2261999
references: Arch Pharm (Weinheim). 2011 Sep;344(9):557-63
references: 21695711
references: Chembiochem. 2006 Dec;7(12):1899-908
references: 17051653
references: Eur J Biochem. 1971 Nov 11;23(1):178-84
references: 5127383
references: J Bacteriol. 2005 Aug;187(15):5067-74
references: 16030198
references: Biochem Biophys Res Commun. 2005 Dec 9;338(1):223-9
references: 16169514
references: Cell Biochem Biophys. 2006;44(3):530-8
references: 16679541
references: Biochemistry. 1996 Jan 9;35(1):160-70
references: 8555170
references: Proc Natl Acad Sci U S A. 2008 May 27;105(21):7347-52
references: 18492808
references: J Bacteriol. 2001 Jun;183(12):3689-703
references: 11371533
references: Methods Enzymol. 1990;188:89-95
references: 2280721
references: FEBS J. 2006 Jul;273(13):2963-76
references: 16734718
references: J Biol Chem. 1990 Apr 15;265(11):6301-11
references: 2156846
references: Biosci Biotechnol Biochem. 2012;76(3):567-74
references: 22451401
references: Crit Rev Biochem Mol Biol. 2006 Jul-Aug;41(4):241-67
references: 16849108
references: Biochemistry. 2006 Jan 24;45(3):1009-16
references: 16411777
references: Biochem J. 2005 Mar 1;386(Pt 2):305-14
references: 15479158
references: Biochem J. 2009 Mar 1;418(2):403-11
references: 18973472
references: Arch Microbiol. 2005 Feb;183(2):130-9
references: 15650824
references: Biochemistry. 2012 Jan 10;51(1):257-64
references: 22122511
references: J Biol Chem. 1998 Dec 25;273(52):34887-95
references: 9857017
references: J Ind Microbiol Biotechnol. 1999 Oct;23(4-5):364-373
references: 11423957
references: J Microbiol. 2004 Jun;42(2):152-5
references: 15357311
references: J Bacteriol. 2004 Aug;186(15):4951-9
references: 15262932
references: J Am Chem Soc. 2005 Dec 7;127(48):16882-91
references: 16316234
references: J Phys Chem A. 2010 Nov 25;114(46):12299-304
references: 21028773
references: Curr Opin Biotechnol. 2011 Jun;22(3):394-400
references: 21071202
references: Chemosphere. 2007 Aug;68(11):2080-4
references: 17368726
references: FEMS Microbiol Lett. 2007 Sep;274(2):323-8
references: 17645527
references: Biochemistry. 1972 Aug 15;11(17):3212-9
references: 5048285
references: Biochemistry. 2004 Oct 26;43(42):13390-6
references: 15491145
references: Appl Environ Microbiol. 1999 Mar;65(3):946-50
references: 10049846
references: J Bacteriol. 2009 Nov;191(21):6758-68
references: 19717587
references: Chem Rev. 2005 Aug;105(8):2999-3093
references: 16092826
references: Biochim Biophys Acta. 1970 Nov 11;220(2):224-38
references: 5487881
references: J Bacteriol. 1997 Oct;179(19):6145-53
references: 9324265