Factors that affect oxygen activation and coupling of the two redox cycles in the aromatization reaction catalyzed by NikD, an unusual amino acid oxidase.
NikD is a flavoprotein oxidase that catalyzes the oxidation of piperideine-2-carboxylate (P2C) to picolinate in a remarkable aromatization reaction comprising two redox cycles and at least one isomerization step. Tyr258 forms part of an "aromatic cage" that surrounds the ring in picolinate and its precursors. Mutation of Tyr258 to Phe does not perturb the structure of nikD but does affect the coupling of the two redox cycles and causes a 10-fold decrease in turnover rate. Tyr258Phe catalyzes a quantitative two-electron oxidation of P2C, but only 60% of the resulting dihydropicolinate intermediate undergoes a second redox cycle to produce picolinate. The mutation does not affect product yield with an alternate substrate (3,4-dehydro-L-proline) that is aromatized in a single two-electron oxidation step. Wild-type and mutant enzymes exhibit identical rate constants for oxidation of P2C to dihydropicolinate and isomerization of a reduced enzyme.dihydropicolinate complex. The observed rates are 200- and 10-fold faster, respectively, than the mutant turnover rate. Release of picolinate from Tyr258Phe is 100-fold faster than turnover. The presence of a bound substrate or product is a key factor in oxygen activation by wild-type nikD, as judged by the 10-75-fold faster rates observed for complexes of the reduced enzyme with picolinate, benzoate, or 1-cyclohexenoate, a 1-deaza-P2C analogue. The reduced Tyr258Phe x 1-cyclohexenoate complex is 25-fold less reactive with oxygen than the wild-type complex. We postulate that mutation of Tyr258 causes subtle changes in active site dynamics that promote release of the reactive dihydropicolinate intermediate and disrupt the efficient synchronization of oxygen activation observed with wild-type nikD.
Kommoju
Phaneeswara-Rao
PR
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, USA.
Bruckner
Robert C
RC
Ferreira
Patricia
P
Carrell
Christopher J
CJ
Mathews
F Scott
FS
Jorns
Marilyn Schuman
MS
D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D018360:Crystallography, X-Ray; D005420:Flavoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism*; D010649:Phenylalanine / Q000235:genetics; D010848:Picolinic Acids / Q000737:chemistry / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology; D014443:Tyrosine / Q000235:genetics
D005420:Flavoproteins; D008024:Ligands; D010848:Picolinic Acids; D014443:Tyrosine; D010649:Phenylalanine; D000594:Amino Acid Oxidoreductases; C030614:picolinic acid; D010100:Oxygen
Clin Microbiol Rev. 1993 Jan;6(1):1-21
8457977
Biochemistry. 2004 Jul 20;43(28):9160-7
15248773
J Biol Chem. 2009 Feb 13;284(7):4392-7
19088070
Biochemistry. 2000 Feb 15;39(6):1515-21
10684633
Arch Biochem Biophys. 1986 May 1;246(2):743-50
3707132
Biochemistry. 2008 Sep 2;47(35):9124-35
18693755
Trends Biochem Sci. 2006 May;31(5):276-83
16600599
Arch Biochem Biophys. 2006 Jul 15;451(2):182-7
16713988
Mol Gen Genet. 2001 Jan;264(5):662-73
11212921
Structure. 1999 Mar 15;7(3):331-45
10368302
J Biol Chem. 1994 Sep 9;269(36):22459-62
8077188
Biochemistry. 2000 Aug 1;39(30):8825-9
10913293
Biochemistry. 2009 Jun 2;48(21):4455-65
19354202
Structure. 2007 Aug;15(8):928-41
17697998
J Am Chem Soc. 2004 Nov 24;126(46):15120-31
15548009
Proc Natl Acad Sci U S A. 2003 Jan 7;100(1):62-7
12506204
J Struct Biol. 1999 Apr-May;125(2-3):156-65
10222271
Biotechnol Lett. 2006 Dec;28(23):1895-900
17043907
Biochemistry. 2005 May 3;44(17):6452-62
15850379
Biochemistry. 2005 Mar 1;44(8):2770-80
15723521
Biochemistry. 2006 May 16;45(19):5985-92
16681370
Methods Enzymol. 1997;276:307-26
27754618
Biochemistry. 2000 Aug 1;39(30):8813-24
10913292
J Biol Chem. 2008 Sep 5;283(36):24738-47
18614534
Gene. 1989 Apr 15;77(1):51-9
2744487
Biochemistry. 2009 Jul 28;48(29):6951-62
19530706
Biochemistry. 1999 Apr 27;38(17):5588-95
10220347
Biochemistry. 2002 Dec 31;41(52):15795-802
12501208
Chem Biol. 2002 Feb;9(2):171-84
11880032
Eur J Biochem. 1998 Jun 1;254(2):347-55
9660190
Acc Chem Res. 2007 May;40(5):325-33
17474709
Biochemistry. 2007 Jan 23;46(3):819-27
17223703
Biochemistry. 2001 Feb 6;40(5):1441-50
11170472
Chemistry. 2009;15(7):1597-603
19130530
Biochem J. 2000 Feb 1;345 Pt 3:487-94
10642506