Site-specific protein labeling methods are highly valuable tools for research and applications. We present a new protein labeling method that allows covalent attachment of a chromo- and fluorogenic flavin (FMN) to any targeted protein using a short flavinylation peptide-tag. We show that this peptide can be as short as 7 residues and can be located at the N-terminus, C-terminus, or in internal regions of the target protein. Analogous to kinase-catalyzed phosphorylation, the flavin is covalently attached via a stable phosphothreonyl linkage. The site-specific covalent tethering of FMN is accomplished by using a bacterial flavin transferase. The covalent coupling of FMN was shown to work in Escherichia coli and Saccharomyces cerevisiae cells and could be performed in vitro, rendering the “Flavin-tag” method a powerful tool for the selective decoration of proteins with a biocompatible redox-active fluorescent chromophore.

As a crucial factor for biocatalysts, protein thermostability often arises from a combination of factors that are often difficult to rationalize. In this work, the thermostable nature of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) was systematically explored using a combinatorial directed evolution approach. For this, a mutagenesis library of HheC mutants was first constructed using error-prone PCR with low mutagenesis frequency. After screening approximately 2000 colonies, six mutants with eight mutation sites were obtained. Those mutation sites were subsequently combined by adopting several rounds of iterative saturation mutagenesis (ISM) approach. After four rounds of saturation mutagenesis, one best mutant ISM-4 with a 3400-fold improvement in half-life (t ) inactivation at 65 °C, 18 °C increase in apparent T value, and 20 °C increase in optimum temperature was obtained, compared to wild-type HheC. To the best of our knowledge, the mutant represents the most thermostable HheC variant reported up to now. Moreover, the mutant was as active as wild-type enzyme for the substrate 1,3-dichloro-2-propanol, and they remained most enantioselectivity of wild-type enzyme in the kinetic resolution of rac-2-chloro-1-phenolethanol, exhibiting a great potential for industrial applications. Our structural investigation highlights that surface loop regions are hot spots for modulating the thermostability of HheC, the residues located at these regions contribute to the thermostability of HheC in a cooperative way, and protein rigidity and oligomeric interface connections contribute to the thermostability of HheC. All of these essential factors could be used for further design of an even more thermostable HheC, which, in turn, could greatly facilitate the application of the enzyme as a biocatalyst.

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Methods for facile site-selective modifications of proteins are in high demand. We have recently shown that a flavin transferase can be used for site-specific covalent attachment of a chromoand fluorogenic flavin (FMN) to any targeted protein. Although this Flavin-tag method resulted in efficient labeling of proteins in vitro, labelling in E. coli cells resulted in partial flavin incorporation. It was also restricted in the type of installed label with only one type of flavin, FMN, being incorporated. Here, we report on an extension of the Flavin-tag method that addresses previous limitations. We demonstrate that co-expression of FAD synthetase improves the flavin incorporation efficiency, allowing complete flavin-labeling of a target protein in E. coli cells. Furthermore, we have found that various flavin derivatives and even a nicotinamide can be covalently attached to a target protein, rendering this method even more versatile and valuable.

Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characteri­zation of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus Mycena chlorophos (McH3H), which catalyzes the con­ver­sion of hispidin to 3-hydroxyhispidin. 3‑Hydroxy­hispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in Escherichia coli and purified by affinity chromato­graphy with a yield of 100 mg/l. McH3H was found to be a single component monomeric NAD(P)H-dependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxy-hispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydro-peroxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin.