FOP:on the (bio)synthesis and biocatalytic applications of the artificial deazaflavin cofactor FO-5′-phosphate

Using enzymes to speed up chemical reactions can help make those reactions more environmentally friendly. Some enzymes need small helper molecules, called cofactors, in order to function properly. F420 is one such cofactor. Because it readily donates electrons to other molecules, especially those that do not typically react with other cofactors, F420 could be a more sustainable alternative to chemical processes that have no biological substitute yet. One difficulty of using F420 is that it either needs to be extracted from slow growing microorganisms such as Mycobacterium smegmatis, or needs to be synthesized chemically, both producing only a small amounts. We therefore designed an artificial cofactor, named FO-5’-phosphate (FOP), which mimics the chemical properties of F420 but can be produced much more efficiently.The first step in creating FOP is to chemically assemble its precursor (FO), which can then be converted to FOP using enzymes. In the first study, we used protein engineering to design a mutant enzyme that was capable of carrying out this last step in the process. In the second study, we designed a way to manufacture FOP inside of fast growing E. coli cells, by introducing enzymes from other organisms in this bacterium, and showed that this method was even more efficient than using Mycobacterium smegmatis to produce F420. Lastly, and most importantly, we found that enzymes that are dependent on F420 work just as well with FOP as they do with F420, which indicates that FOP can be a viable and scalable alternative

Natural Flavins:Occurrence, Role, and Noncanonical Chemistry

Flavoproteins are of key importance to all life on earth for both primary and secondary metabolism. Most flavin-dependent enzymes utilize flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as redox cofactor for single-electron and hydride transfer as well as oxidation and oxygenation chemistry at the C4a-locus. Over the last decades, several naturally occurring modified flavins, like 8-formylFAD, F420, and prenylFMN, and covalently bound flavins have been discovered, and were found to further expand the toolbox of flavin chemistry, showcasing extraordinary redox potentials and unprecedented chemistry. Recently, also several examples of “exotic” flavin chemistry, such as N5-oxygenation, have been identified in enzymes that utilize the standard flavins FMN and FAD. It shows that nature has been extremely inventive in exploiting flavins and flavin derivatives as cofactors for an exceptionally wide variety of reactions. Future research will reveal whether other, so far hidden, flavoenzyme-catalyzed chemistries exit.</p

Flavin-Tag:A Facile Method for Site-Specific Labeling of Proteins with a Flavin Fluorophore

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

A Tailor-Made Deazaflavin-Mediated Recycling System for Artificial Nicotinamide Cofactor Biomimetics

[Image: see text] Nicotinamide adenine dinucleotide (NAD) and its 2′-phosphorylated form NADP are crucial cofactors for a large array of biocatalytically important redox enzymes. Their high cost and relatively poor stability, however, make them less attractive electron mediators for industrial processes. Nicotinamide cofactor biomimetics (NCBs) are easily synthesized, are inexpensive, and are also generally more stable than their natural counterparts. A bottleneck for the application of these artificial hydride carriers is the lack of efficient cofactor recycling methods. Therefore, we engineered the thermostable F(420):NADPH oxidoreductase from Thermobifida fusca (Tfu-FNO), by structure-inspired site-directed mutagenesis, to accommodate the unnatural N1 substituents of eight NCBs. The extraordinarily low redox potential of the natural cofactor F(420)H(2) was then exploited to reduce these NCBs. Wild-type enzyme had detectable activity toward all selected NCBs, with K(m) values in the millimolar range and k(cat) values ranging from 0.09 to 1.4 min(–1). Saturation mutagenesis at positions Gly-29 and Pro-89 resulted in mutants with up to 139 times higher catalytic efficiencies. Mutant G29W showed a k(cat) value of 4.2 s(–1) toward 1-benzyl-3-acetylpyridine (BAP(+)), which is similar to the k(cat) value for the natural substrate NADP(+). The best Tfu-FNO variants for a specific NCB were then used for the recycling of catalytic amounts of these nicotinamides in conversion experiments with the thermostable ene-reductase from Thermus scotoductus (TsOYE). We were able to fully convert 10 mM ketoisophorone with BAP(+) within 16 h, using F(420) or its artificial biomimetic FOP (FO-2′-phosphate) as an efficient electron mediator and glucose-6-phosphate as an electron donor. The generated toolbox of thermostable and NCB-dependent Tfu-FNO variants offers powerful cofactor regeneration biocatalysts for the reduction of several artificial nicotinamide biomimetics at both ambient and high temperatures. In fact, to our knowledge, this enzymatic method seems to be the best-performing NCB-recycling system for BNAH and BAPH thus far

Introducing an Artificial Deazaflavin Cofactor in Escherichia coli and Saccharomyces cerevisiae

[Image: see text] Deazaflavin-dependent whole-cell conversions in well-studied and industrially relevant microorganisms such as Escherichia coli and Saccharomyces cerevisiae have high potential for the biocatalytic production of valuable compounds. The artificial deazaflavin FOP (FO-5′-phosphate) can functionally substitute the natural deazaflavin F(420) and can be synthesized in fewer steps, offering a solution to the limited availability of the latter due to its complex (bio)synthesis. Herein we set out to produce FOP in vivo as a scalable FOP production method and as a means for FOP-mediated whole-cell conversions. Heterologous expression of the riboflavin kinase from Schizosaccharomyces pombe enabled in vivo phosphorylation of FO, which was supplied by either organic synthesis ex vivo, or by a coexpressed FO synthase in vivo, producing FOP in E. coli as well as in S. cerevisiae. Through combined approaches of enzyme engineering as well as optimization of expression systems and growth media, we further improved the in vivo FOP production in both organisms. The improved FOP production yield in E. coli is comparable to the F(420) yield of native F(420)-producing organisms such as Mycobacterium smegmatis, but the former can be achieved in a significantly shorter time frame. Our E. coli expression system has an estimated production rate of 0.078 μmol L(–1) h(–1) and results in an intracellular FOP concentration of about 40 μM, which is high enough to support catalysis. In fact, we demonstrate the successful FOP-mediated whole-cell conversion of ketoisophorone using E. coli cells. In S. cerevisiae, in vivo FOP production by SpRFK using supplied FO was improved through media optimization and enzyme engineering. Through structure-guided enzyme engineering, a SpRFK variant with 7-fold increased catalytic efficiency compared to the wild type was discovered. By using this variant in optimized media conditions, FOP production yield in S. cerevisiae was 20-fold increased compared to the very low initial yield of 0.24 ± 0.04 nmol per g dry biomass. The results show that bacterial and eukaryotic hosts can be engineered to produce the functional deazaflavin cofactor mimic FOP

Engineering a Diverse Ligase Toolbox for Peptide Segment Condensation

The substrate profile of peptiligase, a stable enzyme designed for peptide ligation in aqueous environments, was mapped using six different peptide libraries. The most discriminating substrate binding pocket proved to be the first nucleophile binding subsite (S1), which is crucial for the peptide ligation yield. Two important amino acids shaping the S1 pocket are M213 and L208. A site-saturation library of the M213 position yielded two variants with a significantly broadened substrate profile, i.e., M213G and M213P. Next, examination of two libraries with M213G+L208X and M213P+L208X (with X being any proteinogenic amino acid) resulted in a toolbox of enzymes which can accommodate any proteinogenic amino acid in the S1 pocket, except proline. The applicability of a particular enzyme variant in chemoenzymatic peptide synthesis was demonstrated by coupling at the gram scale of two peptide segments to yield exenatide, a 39-mer therapeutic peptide used in the treatment of diabetes type II. The overall yield of 43% is at least 2-fold higher than yields reported for conventional syntheses of exenatide by full solid-phase peptide synthesis; large-scale production costs are expected to be significantly reduced if the enzymatic coupling process is employed to manufacture this peptide.</p

Thermostable D-amino acid decarboxylases derived from Thermotoga maritima diaminopimelate decarboxylase

Diaminopimelate decarboxylases (DAPDCs) are highly selective enzymes that catalyze the common final step in different lysine biosynthetic pathways, i.e. the conversion of meso-diaminopimelate (DAP) to L-lysine. We examined the modification of the substrate specificity of the thermostable decarboxylase from Thermotoga maritima with the aim to introduce activity with 2-aminopimelic acid (2-APA) since its decarboxylation leads to 6-aminocaproic acid (6-ACA), a building block for the synthesis of nylon-6. Structure-based mutagenesis of the distal carboxylate binding site resulted in a set of enzyme variants with new activities toward different D-amino acids. One of the mutants (E315T) had lost most of its activity toward DAP and primarily acted as a 2-APA decarboxylase. We next used computational modeling to explain the observed shift in catalytic activities of the mutants. The results suggest that predictive computational protocols can support the redesign of the catalytic properties of this class of decarboxylating PLP-dependent enzymes

UTF1 is a chromatin-associated protein involved in ES cell differentiation

Embryonic stem (ES) cells are able to grow indefinitely (self-renewal) and have the potential to differentiate into all adult cell types (pluripotency). The regulatory network that controls pluripotency is well characterized, whereas the molecular basis for the transition from self-renewal to the differentiation of ES cells is much less understood, although dynamic epigenetic gene silencing and chromatin compaction are clearly implicated. In this study, we report that UTF1 (undifferentiated embryonic cell transcription factor 1) is involved in ES cell differentiation. Knockdown of UTF1 in ES and carcinoma cells resulted in a substantial delay or block in differentiation. Further analysis using fluorescence recovery after photobleaching assays, subnuclear fractionations, and reporter assays revealed that UTF1 is a stably chromatin-associated transcriptional repressor protein with a dynamic behavior similar to core histones. An N-terminal Myb/SANT domain and a C-terminal domain containing a putative leucine zipper are required for these properties of UTF1. These data demonstrate that UTF1 is a strongly chromatin-associated protein involved in the initiation of ES cell differentiation

Fluid hydration to prevent post-ERCP pancreatitis in average- to high-risk patients receiving prophylactic rectal NSAIDs (FLUYT trial): Study protocol for a randomized controlled trial

Background: Post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP) is the most common complication of ERCP and may run a severe course. Evidence suggests that vigorous periprocedural hydration can prevent PEP, but studies to date have significant methodological drawbacks. Importantly, evidence for its added value in patients already receiving prophylactic rectal non-steroidal anti-inflammatory drugs (NSAIDs) is lacking and the cost-effectiveness of the approach has not been investigated. We hypothesize that combination therapy of rectal NSAIDs and periprocedural hydration would significantly lower the incidence of post-ERCP pancreatitis compared to rectal NSAIDs alone in moderate- to high-risk patients undergoing ERCP. Methods: The FLUYT trial is a multicenter, parallel group, open label, superiority randomized controlled trial. A total of 826 moderate- to high-risk patients undergoing ERCP that receive prophylactic rectal NSAIDs will be randomized to a control group (no fluids or normal saline with a maximum of 1.5 mL/kg/h and 3 L/24 h) or intervention group (lactated Ringer's solution with 20 mL/kg over 60 min at start of ERCP, followed by 3 mL/kg/h for 8 h thereafter). The primary endpoint is the incidence of post-ERCP pancreatitis. Secondary endpoints include PEP severity, hydration-related complications, and cost-effectiveness. Discussion: The FLUYT trial design, including hydration schedule, fluid type, and sample size, maximize its power of identifying a potential difference in post-ERCP pancreatitis incidence in patients receiving prophylactic rectal NSAIDs