MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/v04/MSV000090294/primaryOK200ProteomicsKristin Burnum-JohnsonOrbitrap EclipseAgilent 8890 GC-MSOrbitrap Exploris 480Pseudomonas Putida Kt2440 (ncbitaxon:160488)https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=d5f28a4ca7684963bcd611bde846a53aKristin.Burnum-Johnson@pnnl.govPacific Northwest National LaboratoryMassIVE67MS:1002864 - No post-translational-modifications are included in the identified peptides of this datasetDeciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional β-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modeling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.Initiation of fatty acid biosynthesis in Pseudomonas putida KT2440.McNaught Kevin J KJ, Kuatsjah Eugene E, Zahn Michael M, Prates Érica T ÉT, Shao Huiling H, Bentley Gayle J GJ, Pickford Andrew R AR, Gruber Josephine N JN, Hestmark Kelley V KV, Jacobson Daniel A DA, Poirier Brenton C BC, Ling Chen C, San Marchi Myrsini M, Michener William E WE, Nicora Carrie D CD, Sanders Jacob N JN, Szostkiewicz Caralyn J CJ, Veličković Dušan D, Zhou Mowei M, Munoz Nathalie N, Kim Young-Mo YM, Magnuson Jon K JK, Burnum-Johnson Kristin E KE, Houk K N KN, McGeehan John E JE, Johnson Christopher W CW, Beckham Gregg T GTacido graso, Acp70, Mst26Aa, acidos grasos, DmelCG18284, antimicrobials, FASN2A, Ray, Compounds, postnatal development, type B, antimicrobial agents, A530043O15Rik, growth and development, neutral molecular compounds, mst 355a, Face-1, Grenz, Readability, microbicides, 9130423F15Rik, Anti Mycobacterial Agent, X Radiation, Biotechnologies., Saturated Fatty Acids, echotomography, CI-SDAP, 3, Esterified Fatty Acid, Grenz Rays, Bacteriocide, D030046F19, molecule, 2210401F17Rik, Esterified, molecula, antimicrobial, A, Antimycobacterial Agents, Eubacteria, molecules, CG8982, enzymes, Antibiotika, 2610003B19Rik, Esterified Fatty, Aliphatic, Anti Bacterial Compound, alanina, Molekuel, X, Gaps, SP, Anti-Bacterial Compounds, Bacteria Woese et al. 2024, Grenz Ray, Aliphatic Acids, X Rays, SDAP, ultrasonic diagnosis, Bacteria <bacteria>, Prokaryotae, ACP, Acp, Antimycobacterial Agent, CG7776, radionuclide imaging, Procaryotae, lipodystrophy, antibiotic, DmelCG17673, decarboxylation, Radiation, FACE1, mutagenesis, fatty acid anabolism, e(Pc), Fettsaeuren, Antibacterial Agent, fatty acid formation, L[[3]]CP3, Acp26Aab, BEST:GH10507, Abufène, shortened, X Ray, Prgs, mandibuloacral dysplasia with type B lipodystrophy, microbicide, monoamino-carboxy-dihydro-borane, prokaryotes, Anti Bacterial Agent, Bacteriocides, Anti-Mycobacterial Agent, Antimycobacterial, Acid, Fatty Acid, DmelCG7776, Saturated Fatty Acid, Antibacterial, Engineerings, MANDIBULOACRAL dysplasia with type B lipodystrophy, initiation, Aliphatic Acid, DmelCG8982, Antibacterial Agents, fatty acids, Agents, DmelLcp3, E(PC), Xrays, BG:DS02740.11, anon-48Ac, Biocatalyst, fatty acid biosynthesis, short, Xray, Prokaryota, Saturated Fatty, GH05739, Esterified Fatty Acids, Ovulin, X-ray image, antibiotique, 8kDa, Biocatalysts, Acp-70A, L Isomer, eubacteria, Agent, Spg70, III, acides gras, GH14582, Roentgen Rays, MADB, Antibiotic, Bacteriocidal, CG7157, Bacteriobiota, roentgenography, antibiotics, DmelCG2043, X-Radiation, Fatty Acids, NADH-ubiquinone oxidoreductase 9.6 kDa subunit, lcp3, radioisotope scanning, LCP-3, Anti Bacterial Compounds, stubby, associated with Mandibuloacral dysplasia, Anti-Mycobacterial, Monera, Pseudomonas putida str. KT2440, Bacteria (ex Cavalier-Smith 1987), Alanine, Anti-Bacterial, Enzyme, CP3, Anti-Mycobacterial Agents, radiography, Crystallographies, fatty acid synthesis, Antibiotics, fungi, ALA, X-ray diagnosis, Antibiotikum, Acp36D, DmelCG7157, mst355a, bacteria, Saturated, ultrasonography, PapB, LcpIII, Rays, L Alanine, enzyme activity, L-Alanine, CT22109, L-Isomer, Roentgen Ray, Anti Mycobacterial Agents, Mst26A, 2-Aminopropionic acid, development, Fettsaeure, echography, Mutageneses, Roentgen, Bacteriocidal Agents, LCP3, L-Isomer Alanine, anon-WO0140519.55, Decarboxylations, (L)-Alanine, Anti Bacterial Agents, distinct, DMLCP3, l(2)28-28-12, postnatal growth, anon-35Fa, 2-Aminopropanoic acid, acide gras, Alanin, Understanding, Ste24p, msP355a, X-ray, 2310039H15Rik, Acp26A, Bacteriocidal Agent, Pseudomonas putida (strain KT2440), Anti-Bacterial Agent, DmelCG5861, Compound, anon-35Fc, Acp70a, prokaryote, ultrasound, DrmSP, X-Radiations, CG2043, Anti-Bacterial Compound, X-Ray, growth, CG17673, Acp26a, STE24Pseudomonas convexa, Pseudomanas putida, DmelCG17673, Bacillus fluorescens putidus, Acp36D, DmelCG7157, mst355a, Bru, Pseudomonas sp. KDB25, Acp70, Mst26Aa, Pseudomonas ovalis, PapB, Ovulin, Raw, DmelCG18284, FASN2A, 8kDa, Acp-70A, Pseudomonas eisenbergii, CT22109, mst 355a, Bacillus putidus, Acp26Aab, BEST:GH10507, Mst26A, Spg70, Pseudomonas arvilla, 9130423F15Rik, Arthrobacter siderocapsulatus, CI-SDAP, Del(8)44H, CG7157, Acp26a., 2210401F17Rik, anon-WO0140519.55, Svc, NADH-ubiquinone oxidoreductase 9.6 kDa subunit, Col4a-1, CG8982, 2610003B19Rik, Pseudomonas striata, msP355a, DmelCG8982, SP, 2310039H15Rik, Acp26A, Pseudomonas incognita, SDAP, Acp70a, ACP, Acp, DrmSP, Pseudomonas rugosa, CG17673fatty acid anabolism, Pseudomonas putida str. KT2440, Pseudomonas putida (strain KT2440)., initiation, fatty acid synthesis, fatty acid biosynthesis, fatty acid formationManuscripts., dental and eye anomalies, Bru, Mass Spectrometry Gas Chromatography, Raw, determination, DNA Proteins, Recombinant DNA, Gas Liquid Chromatography Mass Spectrometry, Proteins, Gas-Liquid-Mass Spectrometry, Petty Laxova Wiedemann syndrome, patent ductus arteriosus, Spectrum Analysis, GCMS, Nomenclature, 2610019N19Rik, Spectrometry-Gas Chromatography, Protein, chemical analysis, Mass, hypertrichosis, PTGFB, Recombinant Protein, Del(8)44H, Gas, PLAB, Biosynthetic, Mass Spectrometry-Gas, Gas-Liquid Chromatography-Mass Spectrometry, Svc, DNA Recombinant, PFD1, Col4a-1, CG6482, Recombinant Proteins, MIC1, Gas Liquid Mass Spectrometry, Mass-Gas Chromatography, Spectrometry, PDF, DmelCG31278, MIC-1, Spectrometries, Gas Chromatography Mass Spectrometry, Biosynthetic Proteins, craniofacial dysostosis, Gas Chromatography-Mass, sample population, Recombinant, Recombinant DNA Proteins, Gas Mass Spectrometry, DNA Recombinant Proteins, Gas-Liquid, Mass Gas Chromatography, hypoplasia of labia majora, Gas-Liquid Chromatography-Mass, sample, Chromatography, Chromatography-Mass Spectrometry, GDF-15, DmelCG31373, NAG-1, assay, DNA, associated, Gas-Mass Spectrometry, Mass Spectrometry-Gas Chromatography, Biosynthetic ProteinfalseRaw data for the characterization of a Pseudomonas putida malonyl-ACP decarboxylaseRaw data for top down LCMS for detecting modifications on recombinant proteins, and GCMS for fatty acid methyl ester analysis. Sample nomenclature is summarized in the Powerpoint and PDF files. Details of experiments are given in the associated manuscript.MSV00009029436796578