Structural highlights
FunctionPublication Abstract from PubMedMicrobial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially-relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of these enzymes their large-scale production in active, soluble, form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the soluble expression of TrzN, an enzyme with environmental applications from Arthrobacter aurescens that catalyzes the hydrolysis of triazine herbicides, in Escherichia coli. Using a combination of X-ray crystallography, kinetic analysis and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme, rather than the metal-ion bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble protein production without increasing the stability of the final, active, form of the enzyme. This study provides a molecular understanding of the importance of the stability metal-ion free states to the accumulation of soluble protein and that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently. Apoenzyme stabilization results in 300-fold increased soluble production of the Zn2+-dependent dechlorinase TrzN.,Jackson CJ, Coppin CW, Carr PD, Aleksandrov A, Wilding M, Sugrue E, Ubels J, Paks M, Newman J, Peat TS, Russell RJ, Field M, Weik M, Oakeshott JG, Scott C Appl Environ Microbiol. 2014 Apr 25. PMID:24771025[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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