Characterization of a thermostable methylaspartate ammonia lyase from Carboxydothermus hydrogenoformans

Hans Raj, Vinod Puthan Veetil, Wiktor Szymanski, Frank J. Dekker, Wim J. Quax, Ben L. Feringa, Dick B. Janssen, Gerrit J. Poelarends*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

Methylaspartate ammonia lyase (MAL; EC 4.3.1.2) catalyzes the reversible addition of ammonia to mesaconate to give (2S,3S)-3-methylaspartate and (2S,3R)-3-methylaspartate as products. MAL is of considerable biocatalytic interest because of its potential use for the asymmetric synthesis of substituted aspartic acids, which are important building blocks for synthetic enzymes, peptides, chemicals, and pharmaceuticals. Here, we have cloned the gene encoding MAL from the thermophilic bacterium Carboxydothermus hydrogenoformans Z-2901. The enzyme (named Ch-MAL) was overproduced in Escherichia coli and purified to homogeneity by immobilized metal affinity chromatography. Ch-MAL is a dimer in solution, consisting of two identical subunits (similar to 49 kDa each), and requires Mg2+ and K+ ions for maximum activity. The optimum pH and temperature for the deamination of (2S,3S)-3-methylaspartic acid are 9.0 and 70A degrees C (k (cat) = 78 s(-1) and K (m) = 16 mM). Heat inactivation assays showed that Ch-MAL is stable at 50A degrees C for > 4 h, which is the highest thermal stability observed among known MALs. Ch-MAL accepts fumarate, mesaconate, ethylfumarate, and propylfumarate as substrates in the ammonia addition reaction. The enzyme also processes methylamine, ethylamine, hydrazine, hydroxylamine, and methoxylamine as nucleophiles that can replace ammonia in the addition to mesaconate, resulting in the corresponding N-substituted methylaspartic acids with excellent diastereomeric excess (> 98% de). This newly identified thermostable MAL appears to be a potentially attractive biocatalyst for the stereoselective synthesis of aspartic acid derivatives on large (industrial) scale.

Original languageEnglish
Pages (from-to)385-397
Number of pages13
JournalApplied Microbiology and Biotechnology
Volume94
Issue number2
DOIs
Publication statusPublished - Apr-2012

Keywords

  • Methylaspartate ammonia lyase
  • Thermostable enzyme
  • Carboxydothermus hydrogenoformans
  • Enzyme catalysis
  • Amino acids
  • CRYSTALLINE 3-METHYLASPARTASE
  • FACULTATIVE ANAEROBE
  • BETA-METHYLASPARTASE
  • ESCHERICHIA-COLI
  • ACID-DERIVATIVES
  • ASPARTIC ACIDS
  • FINE CHEMICALS
  • STRAIN YG-1002
  • BIOCATALYSIS
  • PROTEIN

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