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Characterization and biocatalytic applications of aspartate and methylaspartate ammonia lyases

13 July 2012

PhD ceremony: Mr. V. Puthan Veetil, 16.15 uur, Academiegebouw, Broerstraat 5, Groningen

Dissertation: Characterization and biocatalytic applications of aspartate and methylaspartate ammonia lyases

Promotor(s): prof. W.J. Quax

Faculty: Mathematics and Natural Sciences

In his PhD work, Vinod Puthan Veetil unraveled the catalytic mechanism of aspartate ammonia lyase, which had for long eluded enzymologists, and exploited the biocatalytic potential of an engineered variant of methylaspartate ammonia lyase for the asymmetric synthesis of N-substituted-L-aspartic acids

Aspartate ammonia lyases (aspartases) play an important role in microbial nitrogen metabolism by catalyzing the reversible deamination of L-aspartate to yield fumarate and ammonia. Aspartases are among the most specific enzymes known and, for decades, the high specificity and catalytic activity of these enzymes have been exploited for the commercial production of L-aspartic acid. Although aspartases were first reported as early as 1929, and the first detailed studies were reported in the 1950’s, their exact catalytic mechanism was still unknown.

In his PhD work, Vinod Puthan Veetil has finally unraveled the catalytic mechanism of aspartase. By focusing on the aspartase (AspB) from the thermophilic bacterium Bacillus species YM55-1, he applied site-directed mutagenesis, kinetic, and inhibition studies to evaluate the role of active-site residues in substrate binding and catalysis. Additionally, the crystal structure of AspB in complex with the natural substrate (L-aspartate) was solved. The structural data revealed that substrate binding induces a large conformational change in a conserved loop which brings key catalytic residues in close proximity to the substrate. A catalytic mechanism for AspB has been proposed and the results have provided credence for the notion that AspB and other members of the aspartase/fumarase superfamily use a common substrate binding mode and catalytic mechanism. Furthermore, the findings set the stage for mechanism-based engineering of aspartase for synthesis of aspartic acid derivatives.

In his thesis, Vinod Puthan Veetil also reports the biocatalytic synthesis of a large variety of N-substituted aspartic acids using an engineered variant of the enzyme methylaspartate ammonia lyase. N-substituted aspartic acids are important building blocks for synthetic peptides, nutraceuticals and pharmaceuticals, and for such applications optical purity is paramount. The engineered enzyme indeed catalyzes additions of various structurally diverse amines to fumaric acid with very high enantioselectivity, yielding only the desired L-enantiomers of the amino acid products.

Last modified:15 September 2017 3.42 p.m.
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