Abstract
Heterocycles are important building blocks in pharmaceutical drugs and their enzymatic synthesis is attracting increasing interest. In recent years, various enzymes of the amidohydrolase superfamily were reported to catalyze heterocycle-forming condensation reactions. One of these enzymes, MxcM, is biochemically and kinetically characterized in this study. MxcM generates an imidazoline moiety in the biosynthesis of the natural product pseudochelin A, which features potent anti-inflammatory properties. The enzyme shows maximal activity at 50 °C and pH 10 as well as a kcat/Km value of 22,932 s−1 M−1 at its temperature optimum. Experimental data suggest that the activity of MxcM does not depend on a catalytic metal ion, which is uncommon among amidohydrolases. MxcM is highly active in diverse organic solvents and concentrated salt solutions. Furthermore, we show that MxcM is also capable to introduce imidazoline rings into derivatives of its natural substrate myxochelin B. Overall, MxcM is a solvent-stable, halotolerant enzyme with promising biochemical and kinetic properties and, in future, might become a valuable biocatalyst for the manufacturing of pharmaceutical drugs.
Highlights
The amidohydrolases constitute a large enzyme family with more than 36,000 members [1]
Several amidohydrolases have been reported that are involved in the biosynthesis of pharmacologically active natural products [6,7,8,9,10]
MxcM was shown to catalyze an intramolecular condensation of the β-aminoethyl amide moiety in myxochelin B, thereby generating the characteristic imidazoline moiety of pseudochelin A (Figure 1C)
Summary
The amidohydrolases constitute a large enzyme family with more than 36,000 members [1]. They feature a common (β/α)8 -barrel structural fold and typically possess a metal center, which is required for the activation of a water molecule [2,3,4]. The amidohydrolases CyrG and CyrH were proposed to assemble an uracil moiety in cylindrospermopsin biosynthesis [9]. Another more distantly related homolog, MxcM, was discovered in the pseudochelin biosynthetic gene cluster of the marine bacterium Pseudoalteromonas piscicida S2040 [10]. MxcM was shown to catalyze an intramolecular condensation of the β-aminoethyl amide moiety in myxochelin B, thereby generating the characteristic imidazoline moiety of pseudochelin A (Figure 1C)
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