title: Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea.
abstract: Nitrosomonas europaea is an aerobic nitrifying bacterium that oxidizes ammonia (NH3) to nitrite (NO2 (-)) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4 (+)-dependent O2 uptake by N. europaea by 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and matrix-assisted laser desorption ionization-tandem time of flight (MALDI-TOF/TOF) analyses also confirmed that the fluorescent 28-kDa polypeptide was AmoA.
authors.affiliation: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA.
authors.forename: Kristen
authors.initials: K
authors.lastname: Bennett
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
authors.forename: Natalie C
authors.initials: NC
authors.lastname: Sadler
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA.
authors.forename: Aaron T
authors.initials: AT
authors.lastname: Wright
authors.affiliation: Biosciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico, USA.
authors.forename: Chris
authors.initials: C
authors.lastname: Yeager
authors.affiliation: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina, USA mrhyman@ncsu.edu.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Hyman
mesh_terms: D000332:Aerobiosis; D000641:Ammonia / Q000378:metabolism; D002853:Chromatography, Liquid; D013058:Mass Spectrometry; D043370:Nitrosomonas europaea / Q000737:chemistry / Q000201:enzymology*; D010088:Oxidoreductases / Q000032:analysis*; D010100:Oxygen / Q000378:metabolism; D020543:Proteome / Q000032:analysis*
chemical_list: D020543:Proteome; D000641:Ammonia; D010088:Oxidoreductases; C045582:ammonia monooxygenase; D010100:Oxygen
references.citation: J Bacteriol. 1993 Apr;175(8):2436-44
references.pmid: 8468301
references.citation: J Bacteriol. 1995 Sep;177(17):4908-13
references.pmid: 7665467
references.citation: Biochem J. 1983 Apr 15;212(1):31-7
references.pmid: 6870854
references.citation: FEMS Microbiol Lett. 2017 May 1;364(10):
references.pmid: 28379342
references.citation: Appl Environ Microbiol. 1993 Aug;59(8):2501-10
references.pmid: 16349014
references.citation: J Biol Chem. 1992 Jan 25;267(3):1534-45
references.pmid: 1730700
references.citation: Appl Environ Microbiol. 1999 Oct;65(10):4586-93
references.pmid: 10508093
references.citation: Appl Environ Microbiol. 1994 Jun;60(6):1914-20
references.pmid: 16349282
references.citation: Appl Environ Microbiol. 2004 Aug;70(8):4544-50
references.pmid: 15294784
references.citation: Electrophoresis. 1993 Jul;14(7):619-27
references.pmid: 8375353
references.citation: Methods Enzymol. 1995;249:240-83
references.pmid: 7791614
references.citation: Appl Environ Microbiol. 1990 Apr;56(4):1169-71
references.pmid: 2339874
references.citation: J Bacteriol. 1998 Jul;180(13):3353-9
references.pmid: 9642187
references.citation: Bioinformatics. 2008 Jul 1;24(13):1556-8
references.pmid: 18453552
references.citation: Chem Biol. 2007 Sep;14(9):1043-51
references.pmid: 17884636
references.citation: J Bacteriol. 1993 Apr;175(7):1971-80
references.pmid: 8458839
references.citation: J Bacteriol. 1990 Sep;172(9):5368-73
references.pmid: 2394686
references.citation: Appl Environ Microbiol. 1993 Nov;59(11):3728-35
references.pmid: 16349087
references.citation: Appl Environ Microbiol. 1994 Aug;60(8):3033-5
references.pmid: 8085841
references.citation: Crit Rev Biochem Mol Biol. 2003;38(6):471-95
references.pmid: 14695127
references.citation: Biochim Biophys Acta. 1975 Sep 22;403(1):245-55
references.pmid: 1174548
references.citation: Proc Natl Acad Sci U S A. 2009 Feb 10;106(6):1760-5
references.pmid: 19181854
references.citation: Appl Environ Microbiol. 1999 Feb;65(2):632-9
references.pmid: 9925593
references.citation: Microbiol Rev. 1989 Mar;53(1):68-84
references.pmid: 2496288
references.citation: Curr Opin Chem Biol. 2015 Feb;24:139-44
references.pmid: 25531039
references.citation: Appl Environ Microbiol. 1988 Dec;54(12):3187-90
references.pmid: 16347810
references.citation: Nat Methods. 2007 Oct;4(10):822-7
references.pmid: 17901872
references.citation: Appl Environ Microbiol. 2015 Mar;81(6):1942-8
references.pmid: 25576608
references.citation: Appl Environ Microbiol. 1993 Nov;59(11):3718-27
references.pmid: 16349086
references.citation: Environ Sci Technol. 2006 Sep 1;40(17):5435-42
references.pmid: 16999122
references.citation: J Am Chem Soc. 2003 Apr 23;125(16):4686-7
references.pmid: 12696868
references.citation: Biochem J. 1985 May 1;227(3):719-25
references.pmid: 4004794
references.citation: Microbiology (Reading). 2009 Jan;155(Pt 1):279-284
references.pmid: 19118368
references.citation: Appl Environ Microbiol. 2015 Mar;81(6):1966-76
references.pmid: 25576605
references.citation: Biometals. 2010 Aug;23(4):613-22
references.pmid: 20204476
references.citation: J Biol Chem. 1949 Feb;177(2):751-66
references.pmid: 18110453
references.citation: Biochem J. 1996 Jun 15;316 ( Pt 3):825-31
references.pmid: 8670158
references.citation: Appl Environ Microbiol. 2013 Nov;79(21):6544-51
references.pmid: 23956393
references.citation: Biochim Biophys Acta. 1998 Nov 10;1388(2):373-85
references.pmid: 9858770
references.citation: Angew Chem Int Ed Engl. 2009;48(52):9879-83
references.pmid: 19943299