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Characterization of an inducible, membrane-bound iminodiacetate dehydrogenase from Chelatobacter heintzii ATCC 29600

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Abstract

Iminodiacetate (IDA) is a xenobiotic intermediate common to both aerobic and anaerobic metabolism of nitrilotriacetate (NTA). It is formed by either NTA monooxygenase or NTA dehydrogenase. In this paper the detection and characterization of a membrane-bound iminodiacete dehydrogenase (IDA-DH) from Chelatobacter heintzii ATCC 29600 is reported, which oxidizes IDA to glycine and glyoxylate. Out of 15 compounds tested, IDA was the only substrate for the enzyme. Optimum activity of IDA-DH was found at pH 8.5 and 25°C, respectively, and the Km for IDA was found to be 8mM. Activity of the membrane-bound enzyme was inhibited by KCN, antimycine and dibromomethylisopropyl-benzoquinone. When inhibited by KCN IDA-DH was able to reduce the artificial electron acceptor iodonitrotetrazolium (INT). It was possible to extract IDA-DH from the membranes with 2% cholate, to reconstitute the enzyme into soybean phospholipid vesicles and to obtain IDA-DH activity (more than 50% recovery) using ubiquinone Q1 as the intermediate electron carrier and INT as the final electron acceptor. Growth experiments with different substrates revealed that in all NTA-degrading strains tested both NTA monooxygenase and IDA-DH were only expressed when the cells were grown on NTA or IDA. Furthermore, in Cb. heintzii ATCC 29600 growing exponentially on succinate and ammonia, addition of 0.4 g l-1 NTA led to the induction of the two enzymes within an hour and NTA was utilized simultaneously with succinate. The presence of IDA-DH was confirmed in ten different NTA-degrading strains belonging to three different genera.

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Abbreviations

cA:

component A

cB:

component B

DBMIB:

dibromomethylisopropyl-benzoquinone

HEPES:

hydroxyethylpiperazinethanesulfonic acid

IDA:

iminodiacetate, HN(CH2COOH)2

IDA-DH:

iminodiacetate dehydrogenase

INT:

iodonitrotetrazolium chloride

NTA:

nitrilotriacetate, N(CH2COOH)3

NTA-MO:

nitrilotriacetate monooxygenase

PMS:

phenazine methosulphate

SDS-PAGE:

sodium dodecylsulfate polyacrylamide gel electrophoresis

Suc-DH:

succinate dehydrogenase

References

  • Anonymous (1928) International critical tables. McGraw-Hill Book Company, New York

  • Auling G, Busse H-J, Egli T, El-Banna T & Stackebrandt E (1993) Chelatobacter, gen. nov. and Chelatococcus, gen. nov., two novel genera of the alpha subclass of the Proteobacteria to accomodate the Gram-negative, obligately aerobic, nitrilotriacetate (NTA)-utilizing bacteria Chelatobacter heintzii, sp. nov., and Chelatococcus asaccharovorans, sp. nov., System. Appl. Microbiol. (in press)

  • BamforthCW & LargePJ (1977) Solubilization, partial purification, and properties of N-methylglutamate dehydrogenase from Pseudomonas aminovorans. Biochem. J. 161: 357–370

    Google Scholar 

  • BaterAJ & VenablesWA (1977) The characterisation of inducible dehydrogenases specific for the oxidation of D-alanine, allohydroxyproline, choline and sarcosine as peripheral membrane proteins in Pseudomonas aeruginosa. Biochim. Biophys. Acta 468: 209–226

    Google Scholar 

  • BellyRT, LauffJJ & GoodhueCT (1975) Degradation of ethylenediaminetetraacetic acid by microbial populations from an aerated lagoon. Appl. Microbiol. 29: 787–794

    Google Scholar 

  • BoultonCA, HaywoodGW & LargePJ (1980) N-methylglutamate dehydrogenase, a flavoprotein purified from a new pink trimethylamine-utilizing bacterium. J. Gen. Microbiol. 117: 293–304

    Google Scholar 

  • CrippsRE & NobleAS (1973) The metabolism of nitrilotriacetate by a Pseudomonad. Biochem. J. 136: 1059–1068

    Google Scholar 

  • CroftsAR, MeinhardtSW, JonesKR & SnozziM (1983) The role of the quinone pool in the cyclic electron transfer chain of Rhodopseudomons sphaeroides. Biochim. Biophys. Acta 723: 202–218

    Google Scholar 

  • CroftsAR & WraightCA (1983) The electrochemical domain of photosynthesis. Biochim. Biophys. Acta 726: 149–185

    Google Scholar 

  • EgliT, BallyM & UetzT (1990) Microbial degradation of chelating agents used in detergents with special reference to nitrilotriacetic acid (NTA). Biodegradation 1: 121–132

    Google Scholar 

  • EgliT, WeilenmannH-U, El-BannaT & AulingG (1988) Gramnegative, aerobic, nitrilotriactate-utilizing bacteria from wastewater and soil. Syst. Appl. Microbiol. 10: 297–305

    Google Scholar 

  • EpsteinSS (1972) Toxicological and environmental implications on the use of nitrilotriacetic acid as a detergent builder. Int. J. Environ. Stud. 2: 291–311

    Google Scholar 

  • FirestoneMK & TiedjeJM (1978) Pathway of degradation of nitrilotriacetate by a Pseudomonas species. Appl. Environ. Microbiol. 35: 955–961

    Google Scholar 

  • FochtDD & JosephHA (1971) Bacterial degradation of nitrilotriacetic acid. Can. J. Microbiol. 17: 1553–1556

    Google Scholar 

  • HarlowE & LaneD (1988) Antibodies. A Laboratory Manual. Cold Spring Harbour Laboratory, New York

    Google Scholar 

  • IngledewWJ & PooleRK (1984) The respiratory chains of Escherichia coli. Microbiol. Rev. 48: 222–271

    Google Scholar 

  • Jenal-Wanner U (1991) Anaerobic degradation of nitrilotriacetate in a denitrifying bacterium: Purification and characterization of the nitrilotriacetate dehydrogenase/nitrate reductase enzyme complex. Doctoral thesis, ETH No. 9531, Swiss Federal Institute of Technology, Zürich, Switzerland

  • KakiK, YamaguchiH, IguchiY, TeshimaM, ShirakashiT & KuriyamaM (1986) Isolation and characteristics of nitrilotriacetate degrading-bacteria. J. Ferment. Technol. 64: 103–108

    Google Scholar 

  • KasprzakAA, PapasEJ & SteenkampDJ (1983) Identity of the subunits and the stoichiometry of prosthetic groups in trimethylamine dehydrogenase and dimethylamine dehydrogenase. Biochem. J. 211: 535–541

    Google Scholar 

  • KeeseyJ (1987) Biochemica Information. Boehringer Mannheim Biochemicals, Indianapolis

    Google Scholar 

  • Kemmler J (1992) Biochemistry of nitrilotriacetate degradation in the facultatively denitrifying bacterium TE11. Doctoral thesis, No. 9983, Swiss Federal Institute of Technology, Zürich, Switzerland

  • KitaK, VibatCRT, MeinhardtS, GuestJR & GennisRB (1989) One-step purification from Escherichia coli of complex II (Succinate:ubiquinone oxidoreductase) associated with succinate-reducible cytochrome b556. J. Biol. Chem. 264: 2672–2677

    Google Scholar 

  • KnechtR & YoungJY (1986) Liquid chromatographic determination of amino acids after gas-phase hydrolysis and derivatization with (dimethylamino)-azobenzenesulfonyl chloride. Anal. Chem. 58: 2375–2379

    Google Scholar 

  • LaemmliUK (1970) Cleavage of structural proteins during the assembly of bacteriophage T4. Nature 227: 680–685

    Google Scholar 

  • MatsushitaK, NonobeM, ShinagawaE, AdachiO & AmeyamaM (1987) Reconstitution of pyrroloquinoline quinone-dependant d-glucose oxidase respiratory chain of Escherichia coli with cytochrome \(\varpi \) oxidase. J. Bacteriol. 169: 205–209

    Google Scholar 

  • McFetersGA, EgliT, WilbergE, AlderA, SchneiderRP, SnozziM & GigerW (1990) Activity and adaptation of nitrilotriacetate (NTA)-degrading bacteria: field and laboratory studies. Water Res. 24: 875–881

    Google Scholar 

  • MeibergJBM & HarderW (1979) Dimethylamine dehydrogenase from Hyphomicrobium X: purification and some properties of a new enzyme that oxidizes secundary amines. J. Gen. Microbiol. 115: 49–58

    Google Scholar 

  • OlsiewskiPJ, KaczorowskiGJ & WalshC (1980) Purification and properties of d-amino acid dehydrogenase, an inducible membrane-bound iron-sulfur protein from Escherichia coli B. J. Biol. Chem. 255: 4487–4494

    Google Scholar 

  • PennoyerJD, OhnishiT & TrumpowerBL (1988) Purification and properties of succinate-ubiquinone oxidoreductase complex from Paracoccus denitrificans. Biochim. Biophys. Acta 935: 195–207

    Google Scholar 

  • PetersonGL (1979) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochemistry 83: 346–356

    Google Scholar 

  • PickaverAH (1976) The production of N-nitrosoiminodiacetate from nitrilotriacetete and nitrate by microorganism growing in mixed culture. Soil Biol. Biochem. 8: 13–17

    Google Scholar 

  • ReddyTLP & WeberMM (1986) Solubilization, purification and characterization of succinate dehydrogenase from membranes of Mycobacterium phlei. J. Bacteriol. 167: 1–6

    Google Scholar 

  • SchneiderR, ZürcherF, EgliT & HamerG (1989) Ion chromatography method for iminodiacetic acid determination in biological matrices in the presence of nitrilotriacetic acid. J. Chromat. 462: 293–301

    Google Scholar 

  • SchneiderR (1988) Determination of nitrilotriacetate in biological matrices using ion exclusion chromatography. Anal. Biochem. 173: 278–284

    Google Scholar 

  • TiedjeJM (1980) Nitrilotriacetate: Hindsight and gunsight. In: MakiAM, DicksonKL & CairnsJ (Eds) Biotransformation and Fate of Chemicals in the Aquatic Environment (pp 114–119) ASM, Washington

    Google Scholar 

  • TiedjeJM, MasonBB, WarrenCB & MalekEJ (1973) Metabolism of nitrilotriacetate by cells of Pseudomonas species. Appl. Microbiol. 25: 811–818

    Google Scholar 

  • TowbinH, StaechelinT & GordonG (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and applications. Proc. Natl. Acad. Sci. USA 76: 4350–4353

    Google Scholar 

  • TrijbelsF & VogelsGD (1966) Degradation of allantoin by Pseudomonas acidovorans. Biochim. Biophys. Acta 113: 292–301

    Google Scholar 

  • TushurashaviliPR, GavrikovaEV, LendenevAN & VinogradovAD (1985) Studies on the succinate dehydrogenating system. Isolation and properties of the mitochondrial succinate-ubiquinone reductase. Biochim. Biophys. Acta. 809: 145–159

    Google Scholar 

  • Uetz T (1992) Biochemistry of nitrilotriacetate degradation in obligately aerobic, Gram-negative bacteria. Doctoral thesis, No. 9722, Swiss Federal Institute of Technology, Zürich, Switzerland

  • UetzT, SchneiderR, SnozziM & EgliT (1992) Purification and characterization of a two component monooxygenase that hydroxylates nitrilotriacetate (NTA) from Chelatobacter heintzii ATCC 29600. J. Bacteriol. 174: 1179–1188

    Google Scholar 

  • VaradhacharyA & MaloneyPC (1990) A rapid method for reconstitution of bacterial membrane proteins. Mol. Microbiol. 4: 1407–1411

    Google Scholar 

  • WannerU, KemmlerJ, WeilenmannH-U, EgliT, El-BannaT & AulingG (1990) Isolation and growth of a bacterium able to degrade nitrilotriacetate (NTA) under denitrifying conditions. Biodegradation 1: 31–41

    Google Scholar 

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Authors and Affiliations

  1. Federal Institute for Water Resources and Water Pollution Control (EAWAG), CH-8600, Dübendorf, Switzerland

    Thomas Uetz & Thomas Egli

  2. Swiss Federal Institute of Technology, CH-8600, Dübendorf, Switzerland

    Thomas Uetz & Thomas Egli

  3. Department of Microbiology, Biozentrum, University of Basel, CH-4056, Basel, Switzerland

    Thomas Uetz

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  1. Thomas Uetz
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  2. Thomas Egli
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Uetz, T., Egli, T. Characterization of an inducible, membrane-bound iminodiacetate dehydrogenase from Chelatobacter heintzii ATCC 29600. Biodegradation 3, 423–434 (1992). https://doi.org/10.1007/BF00240364

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  • DOI: https://doi.org/10.1007/BF00240364

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