Splicing of eukaryotic pre-mRNA is carried out by the spliceosome, which assembles stepwise on each splicing substrate. This requires the concerted action of snRNPs and non-snRNP accessory proteins, the functions of which are often not well understood. Of special interest are B complex factors that enter the spliceosome prior to catalytic activation and may alter splicing kinetics and splice site selection. One of these proteins is FBP21, for which we identified several spliceosomal binding partners in a yeast-two-hybrid screen, among them the RNA helicase Brr2. Biochemical and biophysical analyses revealed that an intrinsically disordered region of FBP21 binds to an extended surface of the C-terminal Sec63 unit of Brr2. Additional contacts in the C-terminal helicase cassette are required for allosteric inhibition of Brr2 helicase activity. Furthermore, the direct interaction between FBP21 and the U4/U6 di-snRNA was found to reduce the pool of unwound U4/U6 di-snRNA. Our results suggest FBP21 as a novel key player in the regulation of Brr2.

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Directed evolution has helped enzyme engineering to remarkable successes in the past. A main challenge in directed evolution is to find the most suitable starting point, that is, an enzyme that allows maximum "evolvability". Consisting of a synthetic cofactor embedded in a protein scaffold, artificial metalloenzymes (ArMs) are reminiscent of rough-hewn ancestral metalloproteins and thus could provide an evolutionarily clean slate. Here, we report the design and directed evolution of an ArM with peroxidase-like properties based on the nitrobindin variant, NB4. After identifying a suitable artificial metal cofactor, two rounds of directed evolution were sufficient to elevate the ArM's activity to levels akin to those of some natural peroxidases (up to k cat = 14.1 s −1 and k cat /K m = 52,800 M −1 s −1 ). A substitution to arginine in the distal cofactor environment (position 76) was the key to boost the peroxidase activity. Molecular dynamics simulations reveal a remarkable flexibility in the distal site of the NB4 scaffold that is absent in the nitrobindin wildtype and which allows the unrestricted movement of the catalytically important Arg76. In addition to the oxidation of the common redox mediators (ABTS, syringaldehyde, and 2,6-dimethoxyphenol), the ArM proved efficient in the decolorization of three recalcitrant dyes (indigo carmine, reactive blue 19, and reactive black 5) and was amenable to several rounds of ArM recycling.

Enzymatic oxidative decarboxylation is an up‐and‐coming reaction yet lacking efficient screening methods for the directed evolution of decarboxylases. Here, we describe a simple photoclick assay for the detection of decarboxylation products and its application in a proof‐of‐principle directed evolution study on the decarboxylase OleT. The assay was compatible with two frequently used OleT operation modes (directly using hydrogen peroxide as the enzyme's co‐substrate or using a reductase partner) and the screening of saturation mutagenesis libraries identified two enzyme variants shifting the enzyme's substrate preference from long chain fatty acids toward styrene derivatives. Overall, this photoclick assay holds promise to speed‐up the directed evolution of OleT and other decarboxylases.

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