Protein for the chemoenzymatic production of L-<i>threo</i>-hydroxyaspartate

The invention at hand provides a novel protein for the chemoenzymatic and enantiomerically pure production of L-<i>threo</i>-hydroxyaspartate from L-aspartate.<p>

The naturally occurring protein AsnO (asparagine oxygenase) is part of the CDA biosynthesis gene cluster in Streptomyces coelicor. AsnO is an Fe<sup>2+</sup> and <font face=”symbol”>a</font>-ketoglutarate-dependent hydroxylase. This hydroxylase acts as a building block for non-ribosomally produced CDA (?Calcium-dependent antibiotic?) in vivo. During the catalytic cycle, this class of enzymes couples the oxidative decomposition of <font face=”symbol”>a</font>-ketoglutarate to succinate and CO<sub>2</sub> with the hydroxylation of the substrate (L-asparagine).<p>

In the wild type of AsnO, the side chain of the amino acid residue Asp-241 thereby binds to the NH<sub>2</sub> group of the carboxamide group of L-Asn.
Surprisingly, it was found that directed mutagenesis of Asp-241 to Asn-241 (D241N) results in a binding site for the carboxyl group of an aspartate side chain. By means of this directed mutagenesis, the substrate specificity changes from asparagine to aspartate, and the mutated protein converts aspartate chemoenzymatically to L-threo-hydroxyaspartate in the presence of Fe2+ and a-ketoglutarate.<p>

Thereby, AsnO D241N represents the mutant of the wild type AsnO according to the present invention.

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