300-Fold increase in production of the Zn2+-dependent dechlorinase TrzN in soluble form via apoenzyme stabilization.

Microbial 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 the 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 expression of soluble TrzN, an enzyme from Arthrobacter aurescens with environmental applications 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 of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently.

Ultralow dose CT for follow-up of solid pulmonary nodules: A pilot single-center study using Bland-Altman analysis.

Solid pulmonary nodules are a common finding requiring serial computed tomography (CT) imaging. We sought to explore the detection and measurement accuracy of an ultralow-dose CT (ULDCT) protocol compared with our standard low-dose CT (LDCT) nodule follow-up protocol.In this pragmatic single-center pilot prospective cohort study, patients scheduled for clinically indicated CT surveillance of 1 or more known solid pulmonary nodules >2 mm underwent ULDCT immediately after routine LDCT. The Bland-Altman 95% limits of agreement for diameter and volumetry were calculated.In all, 57 patients underwent 60 imaging episodes, with 170 evaluable nodules. ULDCT detected all known solid pulmonary nodules >2 mm. Bland-Altman analyses demonstrated clinically agreement for both nodule diameter and volume, both of which fell within prespecified limits.This single-center pilot study suggests that ULDCT may be of use in surveillance of known solid pulmonary nodules >2 mm.