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Purification and characterization of β‐methylaspartase from Fusobacterium varium

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Abstract

β‐Methylaspartase (EC 4.3.1.2) was purified 20‐fold in 35% yield from Fusobacterium varium, an obligate anaerobe. The purification steps included heat treatment, fractional precipitation with ammonium sulfate and ethanol, gel filtration, and ion exchange chromatography on DEAE‐Sepharose. The enzyme is dimeric, consisting of two identical 46 kDa subunits, and requires Mg2+ (Km = 0.27 ± 0.01 mM) and K+ (Km = 3.3 ± 0.8 mM) for maximum activity. β‐Methylaspartase‐catalyzed addition of ammonia to mesaconate yielded two diastereomeric amino acids, identified by HPLC as (2S,3S)‐3‐methylaspartate (major product) and (2S,3R)‐3‐methylaspartate (minor product). Optimal activity for the deamination of (2S,3S)‐3‐methylaspartate (Km = 0.51 ± 0.04 mM) was observed at pH 9.7. The N‐terminal protein sequence (30 residues) of the F. varium enzyme is 83% identical to the corresponding sequence of the clostridial enzyme.

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References

  1. Eigen M: Proton transfer, acid-base catalysis, and enzymatic hydrolysis. Angew Chem Int Ed Engl 3: 1–19, 1964

    Article  Google Scholar 

  2. Albery WJ: Application of the Marcus relation to concerted proton transfers. J Chem Soc Faraday Trans 1 78: 1579–1590, 1982

    Article  Google Scholar 

  3. Chiang Y, Kresge AJ, Santaballa JA, Wirz J: Ketonization of acetophenone enol in aqueous buffer solutions. Rate-equilibrium relations and mechanism of the ‘uncatalyzed’ reaction. J Am Chem Soc 110: 5506–5510, 1988

    Article  CAS  Google Scholar 

  4. Kresge AJ: Generation and study of enols and other reactive species. Pure Appl Chem 63: 213–221, 1991

    Article  CAS  Google Scholar 

  5. Amyes TL, Richard JP: Determination of the pK a of ethyl acetate: Brønsted correlation for deprotonation of a simple oxygen ester in aqueous solution. J Am Chem Soc 118: 3129–3141, 1996

    Article  CAS  Google Scholar 

  6. Barker HA, Smyth RD, Wilson RM, Weissbach H: The purification and properties of β-methylaspartase. J Biol Chem 234: 320–328, 1959

    CAS  PubMed  Google Scholar 

  7. Botting NP, Akhtar M, Archer CH, Cohen MA, Thomas NR, Goda S, Minton NP, Gani D: Catalytic mechanism and active site structure of methylaspartate ammonia-lyase: Possible involvement of an electrophilic dehydroalanine reaction centre. In: S.M. Roberts (ed). Molecular Recognition: Chemical and Biochemical Problems II. RSC Special Publication 111, Royal Society of Chemistry, London, 1992, pp 95–109

    Google Scholar 

  8. Pollard JR, Richardson S, Akhtar M, Lasry P, Neal T, Botting NP, Gani D: Mechanism of 3-methylaspartase probed using deuterium and solvent isotope effects and active-site directed reagents: Identification of an essential cysteine residue. Bioorg Med Chem 7: 949–975, 1999

    Article  CAS  PubMed  Google Scholar 

  9. Gani D, Archer CH, Botting NP, Pollard JR: The 3-methylaspartase reaction probed using 2H-and 15N-isotope effects for three substrates: A flip from a concerted to a carbocationic amino-enzyme elimination mechanism upon changing the C-3 stereochemistry in the substrate from R to S. Bioorg Med Chem 7: 977–990, 1999

    Article  CAS  PubMed  Google Scholar 

  10. Wickner RB: Dehydroalanine in histidine ammonia-lyase. J Biol Chem 244: 6550–6552, 1969

    CAS  PubMed  Google Scholar 

  11. Consevage MW, Phillips AT: Presence and quantity of dehydroalanine in histidine ammonia-lyase from Pseudomonas putida. Biochemistry 24: 301–308, 1985

    Article  CAS  PubMed  Google Scholar 

  12. Langer M, Reck G, Reed J, Rétey J: Identification of serine 143 as the most likely precursor of dehydroalanine in the active site of histidine ammonia-lyase. A study of the overexpressed enzyme by site-directed mutagenesis. Biochemistry 33: 6462–6467, 1994

    Article  CAS  PubMed  Google Scholar 

  13. Langer M, Lieber A, Rétey J: Histidine ammonia-lyase mutant S143C is post-translationally converted into fully active wild-type enzyme. Evidence for serine 143 to be the precursor of active site dehydroalanine. Biochemistry 33: 14034–14038, 1994

    Article  CAS  PubMed  Google Scholar 

  14. Taylor RG, McInnes RR: Site-directed mutagenesis of conserved serines in rat histidase. Identification of serine 254 as an essential active site residue. J Biol Chem 269: 27473–27477, 1994

    CAS  PubMed  Google Scholar 

  15. Hodgins DS: Yeast phenylalanine ammonia-lyase. Purification, properties, and the identification of a catalytically essential dehydroalanine. J Biol Chem 246: 2977–2985, 1971

    CAS  PubMed  Google Scholar 

  16. Schuster B, Rétey J: Serine 202 is the putative precursor of the active site dehydroalanine of phenylalanine ammonia-lyase. Site-directed mutagenesis studies on the enzyme from parsley (Petroselinum crispum L.). FEBS Lett 349: 252–254, 1994

    Article  CAS  PubMed  Google Scholar 

  17. Schuster B, Rétey J: The mechanism of action of phenylalanine ammonia-lyase: The role of prosthetic dehydroalanine. Proc Natl Acad Sci USA 92: 8433–8437, 1995

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Langer B, Röther D, Rétey J: Identification of essential amino acids in phenylalanine ammonia-lyase by site-directed mutagenesis. Biochemistry 36: 10867–10871, 1997

    Article  CAS  PubMed  Google Scholar 

  19. Schwede TF, Rétey J, Schulz GE: Crystal structure of histidine ammonia-lyase revealing a novel polypeptide modification as the catalytic electrophile. Biochemistry 38: 5355–5361, 1999

    Article  CAS  PubMed  Google Scholar 

  20. Röther D, Merkel D, Rétey J: Spectroscopic evidence for a 4-methylidene imidazol-5-one in histidine and phenylalanine ammonia-lyases. Angew Chem Int Ed 39: 2462–2464, 2000

    Article  Google Scholar 

  21. Shi W, Dunbar J, Jayasekera MMK, Viola RE, Farber GK: The structure of L-aspartate ammonia-lyase from Escherichia coli. Biochemistry 36: 9136–9144, 1997

    Article  CAS  PubMed  Google Scholar 

  22. Bear M-M, Cammas S, Langlois V, Guérin P: Chemoenzymatic synthesis of poly[(2R,3S)-benzyl β-3-methylmalate]: β-Methylaspartase as a versatile enzyme in the preparation of the chiral precursor. C R Acad Sci Paris (11B) 325: 165–172, 1997

    CAS  Google Scholar 

  23. Archer CH, Gani D: Kinetics and mechanism of syn-elimination of ammonia from (2S,3R)-3-methylaspartic acid by methylaspartase. J C S Chem Commun: 140–142, 1993

  24. Archer CH, Thomas NR, Gani D: Syntheses of (2S,3R)-and (2S,3R)[3-2H]-3-methylaspartic acid: Slow substrates for a syn-elimination reaction catalyzed by methylaspartase. Tetrahedron: Asymmetry 4: 1141–1152, 1993

    Article  CAS  Google Scholar 

  25. van der Werf MJ, van den Tweel WJJ, Kamphuis J, Hartmans S, de Bont JAM: The potential of lyases for the industrial production of optically active compounds. Trends Biotechnol 12: 95–103, 1994

    Article  CAS  PubMed  Google Scholar 

  26. Barker HA, Smyth RD, Wawszkiewicz EJ, Lee MN, Wilson, RM: Enzymatic preparation and characterization of an α-L-β-methylaspartic acid. Arch Biochem Biophys 78: 468–476, 1958

    Article  CAS  PubMed  Google Scholar 

  27. Winkler MF, Williams VR: New substrates for β-methylaspartase. Biochem Biophys Acta 146: 287–289, 1967

    CAS  PubMed  Google Scholar 

  28. Akhtar M, Cohen MA, Gani D: Stereochemical course of the enzymatic amination of chloro-and bromo-fumaric acid by 3-methylaspartate ammonia-lyase. Tetrahedron Lett 28: 2413–2416, 1987

    Article  CAS  Google Scholar 

  29. Akhtar M, Botting NP, Cohen MA, Gani D: Enantiospecific synthesis of 3-substituted aspartic acids via enzymatic amination of substituted fumaric acids. Tetrahedron 43: 5899–5908, 1987

    Article  CAS  Google Scholar 

  30. Asano Y, Kato Y: Occurrence of 3-methylaspartate ammonia-lyase in facultative anaerobes and their application to synthesis of 3-substituted (S)-aspartic acids. Biosci Biotech Biochem 58: 223–224, 1994

    Article  CAS  Google Scholar 

  31. Buckel W, Barker HA: Two pathways of glutamate fermentation by anaerobic bacteria. J Bacteriol 117: 1248–1260, 1974

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Kato Y, Asano Y: 3-Methylaspartate ammonia-lyase as a marker enzyme of the mesaconate pathway for (S)-glutamate fermentation in Enterobacteriaceae. Arch Microbiol 168: 457–463, 1997

    Article  CAS  PubMed  Google Scholar 

  33. Goda, SK, Minton NP, Botting NP, Gani D: Cloning, sequencing, and expression in Escherichia coli of the Clostridium tetanomorphum gene encoding β-methylaspartase and characterization of the recombinant protein. Biochemistry 31: 10747–10756, 1992

    Article  CAS  PubMed  Google Scholar 

  34. Williams VR, Traynham JG: β-Methylaspartase of Bacterium cadaveris. Fed Proc Fed Am Soc Exp Biol 21: 247, 1962

    Google Scholar 

  35. Kato Y, Asano Y: Purification and properties of crystalline 3-methylaspartase from two facultative anaerobes, Citrobacter sp. strain YG-0504 and Morganella morganii strain YG-0601. Biosci Biotech Biochem 59: 93–99, 1995

    Article  CAS  Google Scholar 

  36. Kato Y, Asano Y: 3-Methylaspartate ammonia-lyase from a facultative anaerobe, strain YG-1002. Appl Microbiol Biotechnol 43: 901–907, 1995

    Article  CAS  PubMed  Google Scholar 

  37. Kato Y, Asano Y: Cloning, nucleotide sequencing, and expression of the 3-methylaspartate ammonia-lyase gene from Citrobacter amalonaticus strain YG-1002. Appl Microbiol Biotechnol 50: 468–474, 1998

    Article  CAS  PubMed  Google Scholar 

  38. Loesche WJ, Gibbons RJ: Amino acid fermentation by Fusobacterium nucleatum. Arch Oral Biol 13: 191–201, 1968

    Article  CAS  PubMed  Google Scholar 

  39. Finegold SM: Anaerobic gram-negative rods: Bacteroides, Prevotella, Porphyromonas, Fusobacterium, Bilophilia, Sutterella. In: S.L. Gorbach, J.G. Bartlett, N.R. Blacklow (eds). Infectious Diseases. W.B. Saunders Co., Philadelphia, 1998, pp 1904–1915

    Google Scholar 

  40. Bennett KW, Eley A: Fusobacteria: new taxonomy and related diseases. J Med Microbiol 39: 246–254, 1993

    Article  CAS  PubMed  Google Scholar 

  41. Gharbia SE, Shah HN: Pathways of glutamate catabolism among Fusobacterium species. J Gen Microbiol 137: 1201–1206, 1991

    Article  CAS  PubMed  Google Scholar 

  42. White RL, Ramezani M, Gharbia SE, Seth R, Doherty-Kirby AL, Shah HN: Stable-isotope studies of glutamate catabolism in Fusobacterium nucleatum. Biotechnol Appl Biochem 22: 385–396, 1995

    CAS  PubMed  Google Scholar 

  43. Foglesong MA, Cruden DL, Markovetz AJ: Pleomorphism of fusobacteria isolated from the cockroach hindgut. J Bacteriol 158: 474–480, 1984

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Mori K, Nomi H, Chuman T, Kohno M, Kato K, Noguchi M: Synthesis and absolute stereochemistry of serricornin [(4S,6S,7S)-4,6-dimethyl-7-hydroxy-3-nonanone]. Tetrahedron 38: 3705–3711, 1982

    Article  CAS  Google Scholar 

  45. Lam S: Resolution of D-and L-amino acids after precolumn derivatization with o-phthalaldehyde by mixed chelation with Cu(II)-L-proline. J Chromatogr 355: 157–164, 1986

    Article  CAS  Google Scholar 

  46. White RL, Smith KC, DeMarco AC: Biosynthesis of 5-hydroxy-4-oxo-L-norvaline in Streptomyces akiyoshiensis. Can J Chem 72: 1645–1655, 1994

    Article  CAS  Google Scholar 

  47. Botting NP, Akhtar M, Cohen MA, Gani D: Substrate specificity of the 3-methylaspartate ammonia-lyase reaction: Observation of differential relative reaction rates for substrate-product pairs. Biochemistry 27: 2953–2955, 1988

    Article  CAS  PubMed  Google Scholar 

  48. Ramezani M, MacIntosh SE, White RL: Utilization of D-amino acids by Fusobacterium nucleatum and Fusobacterium varium. Amino Acids 17: 185–193, 1999

    Article  CAS  PubMed  Google Scholar 

  49. Wilson KJ, Yuan PM: Protein and peptide purification. In: J.B.C. Findlay, M.J. Geisow (eds). Protein Sequencing: A Practical Approach. Oxford University Press, New York, 1989, p. 13.

    Google Scholar 

  50. Bodanszky M, Marconi GG: Configuration of the β-methylaspartic acid residue in amphomycin. J Antibiot 23: 238–241, 1970

    Article  CAS  PubMed  Google Scholar 

  51. Hanson KR, Havir EA: The enzymatic elimination of ammonia. In: P.D. Boyer (ed). The Enzymes. Vol. VII. Academic Press, New York, 1972, pp 75–166

    Google Scholar 

  52. Takagi JS, Tokushige M, Shimura Y, Kanehisa M: L-Aspartate ammonia-lyase and fumarate hydratase share extensive sequence homology. Biochem Biophys Res Commun 138: 568–572, 1986

    Article  CAS  PubMed  Google Scholar 

  53. Woods SA, Miles JS, Roberts RE, Guest JR: Structural and functional relationships between fumarase and aspartase. Biochem J 237: 547–557, 1986

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Babbitt PC, Hasson MS, Wedekind JE, Palmer DRJ, Barrett WC, Reed GH, Rayment I, Ringe D, Kenyon GL, Gerlt JA: The enolase superfamily: A general strategy for enzyme-catalyzed abstraction of the α-protons of carboxylic acids. Biochemistry 35: 16489–16501, 1996

    Article  CAS  PubMed  Google Scholar 

  55. Williams SE, Woolridge EM, Ransom SC, Landro JA, Babbitt PC, Kozarich JW: 3-Carboxy-cis,cis-muconate lactonizing enzyme from Pseudomonas putida is homologous to the class II fumarase family: A new reaction in the evolution of a mechanistic motif. Biochemistry 31: 9768–9776, 1992

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, B3H 4H7, Canada

    Stephen L. Bearne, Jennifer E. McDonnell & Jesper Grønlund

  2. Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, B3H 4H7, Canada

    Robert L. White & Shervin Bahrami

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  1. Stephen L. Bearne
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Correspondence to Stephen L. Bearne.

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Bearne, S.L., White, R.L., McDonnell, J.E. et al. Purification and characterization of β‐methylaspartase from Fusobacterium varium. Mol Cell Biochem 221, 117–126 (2001). https://doi.org/10.1023/A:1010938111292

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