TY - JOUR
T1 - A New Microbial Pathway for Organophosphonate Degradation Catalyzed by Two Previously Misannotated Non-Heme-Iron Oxygenases
AU - Rajakovich, Lauren J.
AU - Pandelia, Maria Eirini
AU - Mitchell, Andrew J.
AU - Chang, Wei Chen
AU - Zhang, Bo
AU - Boal, Amie K.
AU - Krebs, Carsten
AU - Bollinger, J. Martin
N1 - Funding Information:
The authors thank Prof. Christopher J. Schofield (University of Oxford, Oxford, U.K.) for the library of betaine compounds and the Ps BBOX plasmid, Prof. Wilfred A. van der Donk (University of Illinois at Urbana-Champaign, Urbana, IL) for the library of phosphonate compounds, the Shared Fermentation Facility of The Pennsylvania State University (University Park, PA) for use of the Microfluidics M-110EH-30 micro-fluidizer processor, Mr. Henry Gong at the Pennsylvania State Materials Research Institute (University Park, PA) for ICP-AES analysis, and Dr. Emmanuel Hatzakis at the Pennsylvania State University Nuclear Magnetic Resonance facility. Use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract DE-AC02-06CH11357. GM/CA@APS has been funded in whole or in part with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corp. and Michigan Technology Tri-Corridor Grant 085P1000817.
Funding Information:
@W.-c.C.: Department of Chemistry, North Carolina State University, Raleigh, NC 27695. #B.Z.: REG Life Sciences, LLC, South San Francisco, CA 94080. Funding This work was funded by the National Science Foundation (MCB-1330784 to J.M.B. and C.K.) and the National Institutes of Health (GM119707 to A.K.B., GM111978 to M.-E.P., and GM127079 to C.K.). Notes The authors declare no competing financial interest.
Publisher Copyright:
© 2019 American Chemical Society.
PY - 2019/3/26
Y1 - 2019/3/26
N2 - The assignment of biochemical functions to hypothetical proteins is challenged by functional diversification within many protein structural superfamilies. This diversification, which is particularly common for metalloenzymes, renders functional annotations that are founded solely on sequence and domain similarities unreliable and often erroneous. Definitive biochemical characterization to delineate functional subgroups within these superfamilies will aid in improving bioinformatic approaches for functional annotation. We describe here the structural and functional characterization of two non-heme-iron oxygenases, TmpA and TmpB, which are encoded by a genomically clustered pair of genes found in more than 350 species of bacteria. TmpA and TmpB are functional homologues of a pair of enzymes (PhnY and PhnZ) that degrade 2-aminoethylphosphonate but instead act on its naturally occurring, quaternary ammonium analogue, 2-(trimethylammonio)ethylphosphonate (TMAEP). TmpA, an iron(II)- and 2-(oxo)glutarate-dependent oxygenase misannotated as a γ-butyrobetaine (γbb) hydroxylase, shows no activity toward γbb but efficiently hydroxylates TMAEP. The product, (R)-1-hydroxy-2-(trimethylammonio)ethylphosphonate [(R)-OH-TMAEP], then serves as the substrate for the second enzyme, TmpB. By contrast to its purported phosphohydrolytic activity, TmpB is an HD-domain oxygenase that uses a mixed-valent diiron cofactor to enact oxidative cleavage of the C-P bond of its substrate, yielding glycine betaine and phosphate. The high specificities of TmpA and TmpB for their N-trimethylated substrates suggest that they have evolved specifically to degrade TMAEP, which was not previously known to be subject to microbial catabolism. This study thus adds to the growing list of known pathways through which microbes break down organophosphonates to harvest phosphorus, carbon, and nitrogen in nutrient-limited niches.
AB - The assignment of biochemical functions to hypothetical proteins is challenged by functional diversification within many protein structural superfamilies. This diversification, which is particularly common for metalloenzymes, renders functional annotations that are founded solely on sequence and domain similarities unreliable and often erroneous. Definitive biochemical characterization to delineate functional subgroups within these superfamilies will aid in improving bioinformatic approaches for functional annotation. We describe here the structural and functional characterization of two non-heme-iron oxygenases, TmpA and TmpB, which are encoded by a genomically clustered pair of genes found in more than 350 species of bacteria. TmpA and TmpB are functional homologues of a pair of enzymes (PhnY and PhnZ) that degrade 2-aminoethylphosphonate but instead act on its naturally occurring, quaternary ammonium analogue, 2-(trimethylammonio)ethylphosphonate (TMAEP). TmpA, an iron(II)- and 2-(oxo)glutarate-dependent oxygenase misannotated as a γ-butyrobetaine (γbb) hydroxylase, shows no activity toward γbb but efficiently hydroxylates TMAEP. The product, (R)-1-hydroxy-2-(trimethylammonio)ethylphosphonate [(R)-OH-TMAEP], then serves as the substrate for the second enzyme, TmpB. By contrast to its purported phosphohydrolytic activity, TmpB is an HD-domain oxygenase that uses a mixed-valent diiron cofactor to enact oxidative cleavage of the C-P bond of its substrate, yielding glycine betaine and phosphate. The high specificities of TmpA and TmpB for their N-trimethylated substrates suggest that they have evolved specifically to degrade TMAEP, which was not previously known to be subject to microbial catabolism. This study thus adds to the growing list of known pathways through which microbes break down organophosphonates to harvest phosphorus, carbon, and nitrogen in nutrient-limited niches.
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U2 - 10.1021/acs.biochem.9b00044
DO - 10.1021/acs.biochem.9b00044
M3 - Article
C2 - 30789718
AN - SCOPUS:85062878672
SN - 0006-2960
VL - 58
SP - 1627
EP - 1647
JO - Biochemistry
JF - Biochemistry
IS - 12
ER -