, Volume 3, Issue 4, pp 423–434 | Cite as

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

  • Thomas Uetz
  • Thomas Egli


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.

Key words

membrane protein biodegradation iminodiacetate iminodiacetate dehydrogenase nitrilotriacetate (NTA) ubiquinones 



component A


component B




hydroxyethylpiperazinethanesulfonic acid


iminodiacetate, HN(CH2COOH)2


iminodiacetate dehydrogenase


iodonitrotetrazolium chloride


nitrilotriacetate, N(CH2COOH)3


nitrilotriacetate monooxygenase


phenazine methosulphate


sodium dodecylsulfate polyacrylamide gel electrophoresis


succinate dehydrogenase


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Anonymous (1928) International critical tables. McGraw-Hill Book Company, New YorkGoogle Scholar
  2. 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)Google Scholar
  3. BamforthCW & LargePJ (1977) Solubilization, partial purification, and properties of N-methylglutamate dehydrogenase from Pseudomonas aminovorans. Biochem. J. 161: 357–370Google Scholar
  4. 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: 209–226Google Scholar
  5. BellyRT, LauffJJ & GoodhueCT (1975) Degradation of ethylenediaminetetraacetic acid by microbial populations from an aerated lagoon. Appl. Microbiol. 29: 787–794Google Scholar
  6. BoultonCA, HaywoodGW & LargePJ (1980) N-methylglutamate dehydrogenase, a flavoprotein purified from a new pink trimethylamine-utilizing bacterium. J. Gen. Microbiol. 117: 293–304Google Scholar
  7. CrippsRE & NobleAS (1973) The metabolism of nitrilotriacetate by a Pseudomonad. Biochem. J. 136: 1059–1068Google Scholar
  8. CroftsAR, MeinhardtSW, JonesKR & SnozziM (1983) The role of the quinone pool in the cyclic electron transfer chain of Rhodopseudomons sphaeroides. Biochim. Biophys. Acta 723: 202–218Google Scholar
  9. CroftsAR & WraightCA (1983) The electrochemical domain of photosynthesis. Biochim. Biophys. Acta 726: 149–185Google Scholar
  10. EgliT, BallyM & UetzT (1990) Microbial degradation of chelating agents used in detergents with special reference to nitrilotriacetic acid (NTA). Biodegradation 1: 121–132Google Scholar
  11. EgliT, WeilenmannH-U, El-BannaT & AulingG (1988) Gramnegative, aerobic, nitrilotriactate-utilizing bacteria from wastewater and soil. Syst. Appl. Microbiol. 10: 297–305Google Scholar
  12. EpsteinSS (1972) Toxicological and environmental implications on the use of nitrilotriacetic acid as a detergent builder. Int. J. Environ. Stud. 2: 291–311Google Scholar
  13. FirestoneMK & TiedjeJM (1978) Pathway of degradation of nitrilotriacetate by a Pseudomonas species. Appl. Environ. Microbiol. 35: 955–961Google Scholar
  14. FochtDD & JosephHA (1971) Bacterial degradation of nitrilotriacetic acid. Can. J. Microbiol. 17: 1553–1556Google Scholar
  15. HarlowE & LaneD (1988) Antibodies. A Laboratory Manual. Cold Spring Harbour Laboratory, New YorkGoogle Scholar
  16. IngledewWJ & PooleRK (1984) The respiratory chains of Escherichia coli. Microbiol. Rev. 48: 222–271Google Scholar
  17. 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, SwitzerlandGoogle Scholar
  18. KakiK, YamaguchiH, IguchiY, TeshimaM, ShirakashiT & KuriyamaM (1986) Isolation and characteristics of nitrilotriacetate degrading-bacteria. J. Ferment. Technol. 64: 103–108Google Scholar
  19. KasprzakAA, PapasEJ & SteenkampDJ (1983) Identity of the subunits and the stoichiometry of prosthetic groups in trimethylamine dehydrogenase and dimethylamine dehydrogenase. Biochem. J. 211: 535–541Google Scholar
  20. KeeseyJ (1987) Biochemica Information. Boehringer Mannheim Biochemicals, IndianapolisGoogle Scholar
  21. Kemmler J (1992) Biochemistry of nitrilotriacetate degradation in the facultatively denitrifying bacterium TE11. Doctoral thesis, No. 9983, Swiss Federal Institute of Technology, Zürich, SwitzerlandGoogle Scholar
  22. 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: 2672–2677Google Scholar
  23. KnechtR & YoungJY (1986) Liquid chromatographic determination of amino acids after gas-phase hydrolysis and derivatization with (dimethylamino)-azobenzenesulfonyl chloride. Anal. Chem. 58: 2375–2379Google Scholar
  24. LaemmliUK (1970) Cleavage of structural proteins during the assembly of bacteriophage T4. Nature 227: 680–685Google Scholar
  25. 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: 205–209Google Scholar
  26. McFetersGA, EgliT, WilbergE, AlderA, SchneiderRP, SnozziM & GigerW (1990) Activity and adaptation of nitrilotriacetate (NTA)-degrading bacteria: field and laboratory studies. Water Res. 24: 875–881Google Scholar
  27. MeibergJBM & HarderW (1979) Dimethylamine dehydrogenase from Hyphomicrobium X: purification and some properties of a new enzyme that oxidizes secundary amines. J. Gen. Microbiol. 115: 49–58Google Scholar
  28. 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: 4487–4494Google Scholar
  29. PennoyerJD, OhnishiT & TrumpowerBL (1988) Purification and properties of succinate-ubiquinone oxidoreductase complex from Paracoccus denitrificans. Biochim. Biophys. Acta 935: 195–207Google Scholar
  30. PetersonGL (1979) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochemistry 83: 346–356Google Scholar
  31. PickaverAH (1976) The production of N-nitrosoiminodiacetate from nitrilotriacetete and nitrate by microorganism growing in mixed culture. Soil Biol. Biochem. 8: 13–17Google Scholar
  32. ReddyTLP & WeberMM (1986) Solubilization, purification and characterization of succinate dehydrogenase from membranes of Mycobacterium phlei. J. Bacteriol. 167: 1–6Google Scholar
  33. SchneiderR, ZürcherF, EgliT & HamerG (1989) Ion chromatography method for iminodiacetic acid determination in biological matrices in the presence of nitrilotriacetic acid. J. Chromat. 462: 293–301Google Scholar
  34. SchneiderR (1988) Determination of nitrilotriacetate in biological matrices using ion exclusion chromatography. Anal. Biochem. 173: 278–284Google Scholar
  35. TiedjeJM (1980) Nitrilotriacetate: Hindsight and gunsight. In: MakiAM, DicksonKL & CairnsJ (Eds) Biotransformation and Fate of Chemicals in the Aquatic Environment (pp 114–119) ASM, WashingtonGoogle Scholar
  36. TiedjeJM, MasonBB, WarrenCB & MalekEJ (1973) Metabolism of nitrilotriacetate by cells of Pseudomonas species. Appl. Microbiol. 25: 811–818Google Scholar
  37. TowbinH, StaechelinT & GordonG (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and applications. Proc. Natl. Acad. Sci. USA 76: 4350–4353Google Scholar
  38. TrijbelsF & VogelsGD (1966) Degradation of allantoin by Pseudomonas acidovorans. Biochim. Biophys. Acta 113: 292–301Google Scholar
  39. TushurashaviliPR, GavrikovaEV, LendenevAN & VinogradovAD (1985) Studies on the succinate dehydrogenating system. Isolation and properties of the mitochondrial succinate-ubiquinone reductase. Biochim. Biophys. Acta. 809: 145–159Google Scholar
  40. Uetz T (1992) Biochemistry of nitrilotriacetate degradation in obligately aerobic, Gram-negative bacteria. Doctoral thesis, No. 9722, Swiss Federal Institute of Technology, Zürich, SwitzerlandGoogle Scholar
  41. 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: 1179–1188Google Scholar
  42. VaradhacharyA & MaloneyPC (1990) A rapid method for reconstitution of bacterial membrane proteins. Mol. Microbiol. 4: 1407–1411Google Scholar
  43. 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: 31–41Google Scholar

Copyright information

© Kluwer Academic Publishers 1993

Authors and Affiliations

  • Thomas Uetz
    • 1
    • 3
    • 2
  • Thomas Egli
    • 1
    • 2
  1. 1.Federal Institute for Water Resources and Water Pollution Control (EAWAG)DübendorfSwitzerland
  2. 2.Swiss Federal Institute of TechnologyDübendorfSwitzerland
  3. 3.Department of Microbiology, BiozentrumUniversity of BaselBaselSwitzerland

Personalised recommendations