Understanding and Improving the Activity of Flavin-Dependent Halogenases via Random and Targeted Mutagenesis.

Flavin-dependent halogenases (FDHs) catalyze the halogenation of organic substrates by coordinating reactions of reduced flavin, molecular oxygen, and chloride. Targeted and random mutagenesis of these enzymes have been used to both understand and alter their reactivity. These studies have led to insights into residues essential for catalysis and FDH variants with improved stability, expanded substrate scope, and altered site selectivity. Mutations throughout FDH structures have contributed to all of these advances. More recent studies have sought to rationalize the impact of these mutations on FDH function and to identify new FDHs to deepen our understanding of this enzyme class and to expand their utility for biocatalytic applications.

Protein Evolution and Design.

This article introduces the Protein Evolution and Design theme of the Annual Review of Biochemistry Volume 87.

Myxococcus xanthus encapsulin cargo protein EncD is a flavin-binding protein with ferric reductase activity.

Encapsulins are protein nanocompartments that regulate cellular metabolism in several bacteria and archaea. Myxococcus xanthus encapsulins protect the bacterial cells against oxidative stress by sequestering cytosolic iron. These encapsulins are formed by the shell protein EncA and three cargo proteins: EncB, EncC, and EncD. EncB and EncC form rotationally symmetric decamers with ferroxidase centers (FOCs) that oxidize Fe+2 to Fe+3 for iron storage in mineral form. However, the structure and function of the third cargo protein, EncD, have yet to be determined. Here, we report the x-ray crystal structure of EncD in complex with flavin mononucleotide. EncD forms an α-helical hairpin arranged as an antiparallel dimer, but unlike other flavin-binding proteins, it has no ß-sheet, showing that EncD and its homologs represent a unique class of bacterial flavin-binding proteins. The cryo-EM structure of EncA-EncD encapsulins confirms that EncD binds to the interior of the EncA shell via its C-terminal targeting peptide. With only 100 amino acids, the EncD α-helical dimer forms the smallest flavin-binding domain observed to date. Unlike EncB and EncC, EncD lacks a FOC, and our biochemical results show that EncD instead is a NAD(P)H-dependent ferric reductase, indicating that the M. xanthus encapsulins act as an integrated system for iron homeostasis. Overall, this work contributes to our understanding of bacterial metabolism and could lead to the development of technologies for iron biomineralization and the production of iron-containing materials for the treatment of various diseases associated with oxidative stress.

Deciphering the evolution of flavin-dependent monooxygenase stereoselectivity using ancestral sequence reconstruction.

Controlling the selectivity of a reaction is critical for target-oriented synthesis. Accessing complementary selectivity profiles enables divergent synthetic strategies, but is challenging to achieve in biocatalytic reactions given enzymes' innate preferences of a single selectivity. Thus, it is critical to understand the structural features that control selectivity in biocatalytic reactions to achieve tunable selectivity. Here, we investigate the structural features that control the stereoselectivity in an oxidative dearomatization reaction that is key to making azaphilone natural products. Crystal structures of enantiocomplementary biocatalysts guided the development of multiple hypotheses centered on the structural features that control the stereochemical outcome of the reaction; however, in many cases, direct substitutions of active site residues in natural proteins led to inactive enzymes. Ancestral sequence reconstruction (ASR) and resurrection were employed as an alternative strategy to probe the impact of each residue on the stereochemical outcome of the dearomatization reaction. These studies suggest that two mechanisms are active in controlling the stereochemical outcome of the oxidative dearomatization reaction: one involving multiple active site residues in AzaH and the other dominated by a single Phe to Tyr switch in TropB and AfoD. Moreover, this study suggests that the flavin-dependent monooxygenases (FDMOs) adopt simple and flexible strategies to control stereoselectivity, which has led to stereocomplementary azaphilone natural products produced by fungi. This paradigm of combining ASR and resurrection with mutational and computational studies showcases sets of tools for understanding enzyme mechanisms and provides a solid foundation for future protein engineering efforts.

Pumping iron: A multi-omics analysis of two extremophilic algae reveals iron economy management.

Marine algae are responsible for half of the world's primary productivity, but this critical carbon sink is often constrained by insufficient iron. One species of marine algae, Dunaliella tertiolecta, is remarkable for its ability to maintain photosynthesis and thrive in low-iron environments. A related species, Dunaliella salina Bardawil, shares this attribute but is an extremophile found in hypersaline environments. To elucidate how algae manage their iron requirements, we produced high-quality genome assemblies and transcriptomes for both species to serve as a foundation for a comparative multiomics analysis. We identified a host of iron-uptake proteins in both species, including a massive expansion of transferrins and a unique family of siderophore-iron-uptake proteins. Complementing these multiple iron-uptake routes, ferredoxin functions as a large iron reservoir that can be released by induction of flavodoxin. Proteomic analysis revealed reduced investment in the photosynthetic apparatus coupled with remodeling of antenna proteins by dramatic iron-deficiency induction of TIDI1, which is closely related but identifiably distinct from the chlorophyll binding protein, LHCA3. These combinatorial iron scavenging and sparing strategies make Dunaliella unique among photosynthetic organisms.

Bacterial stigmasterol degradation involving radical flavin delta-24 desaturase and molybdenum-dependent C26 hydroxylase.

Sterols are ubiquitous membrane constituents that persist to a large extent in the environment due to their water insolubility and chemical inertness. Recently, an oxygenase-independent sterol degradation pathway was discovered in a cholesterol-grown denitrifying bacterium Sterolibacterium (S.) denitrificans. It achieves hydroxylation of the unactivated primary C26 of the isoprenoid side chain to an allylic alcohol via a phosphorylated intermediate in a four-step ATP-dependent enzyme cascade. However, this pathway is incompatible with the degradation of widely distributed steroids containing a double bond at C22 in the isoprenoid side chain such as the plant sterol stigmasterol. Here, we have enriched a prototypical delta-24 desaturase from S. denitrificans, which catalyzes the electron acceptor-dependent oxidation of the intermediate stigmast-1,4-diene-3-one to a conjugated (22,24)-diene. We suggest an α4ß4 architecture of the 440 kDa enzyme, with each subunit covalently binding an flavin mononucleotide cofactor to a histidyl residue. As isolated, both flavins are present as red semiquinone radicals, which can be reduced by stigmast-1,4-diene-3-one but cannot be oxidized even with strong oxidizing agents. We propose a mechanism involving an allylic radical intermediate in which two flavin semiquinones each abstract one hydrogen atom from the substrate. The conjugated delta-22,24 moiety formed allows for the subsequent hydroxylation of the terminal C26 with water by a heterologously produced molybdenum-dependent steroid C26 dehydrogenase 2. In conclusion, the pathway elucidated for delta-22 steroids achieves oxygen-independent hydroxylation of the isoprenoid side chain by bypassing the ATP-dependent formation of a phosphorylated intermediate.

Protocatechuate hydroxylase is a novel group A flavoprotein monooxygenase with a unique substrate recognition mechanism.

Para-hydroxybenzoate hydroxylase (PHBH) is a group A flavoprotein monooxygenase that hydroxylates p-hydroxybenzoate to protocatechuate (PCA). Despite intensive studies of Pseudomonas aeruginosa p-hydroxybenzoate hydroxylase (PaPobA), the catalytic reactions of extremely diverse putative PHBH isozymes remain unresolved. We analyzed the phylogenetic relationships of known and predicted PHBHs and identified eight divergent clades. Clade F contains a protein that lacks the critical amino acid residues required for PaPobA to generate PHBH activity. Among proteins in this clade, Xylophilus ampelinus PobA (XaPobA) preferred PCA as a substrate and is the first known natural PCA 5-hydroxylase (PCAH). Crystal structures and kinetic properties revealed similar mechanisms of substrate carboxy group recognition between XaPobA and PaPobA. The unique Ile75, Met72, Val199, Trp201, and Phe385 residues of XaPobA form the bottom of a hydrophobic cavity with a shape that complements the 3-and 4-hydroxy groups of PCA and its binding site configuration. An interaction between the δ-sulfur atom of Met210 and the aromatic ring of PCA is likely to stabilize XaPobA-PCA complexes. The 4-hydroxy group of PCA forms a hydrogen bond with the main chain carbonyl of Thr294. These modes of binding constitute a novel substrate recognition mechanism that PaPobA lacks. This mechanism characterizes XaPobA and sheds light on the diversity of catalytic mechanisms of PobA-type PHBHs and group A flavoprotein monooxygenases.

Complex II ambiguities-FADH2 in the electron transfer system.

The prevailing notion that reduced cofactors NADH and FADH2 transfer electrons from the tricarboxylic acid cycle to the mitochondrial electron transfer system creates ambiguities regarding respiratory Complex II (CII). CII is the only membrane-bound enzyme in the tricarboxylic acid cycle and is part of the electron transfer system of the mitochondrial inner membrane feeding electrons into the coenzyme Q-junction. The succinate dehydrogenase subunit SDHA of CII oxidizes succinate and reduces the covalently bound prosthetic group FAD to FADH2 in the canonical forward tricarboxylic acid cycle. However, several graphical representations of the electron transfer system depict FADH2 in the mitochondrial matrix as a substrate to be oxidized by CII. This leads to the false conclusion that FADH2 from the ß-oxidation cycle in fatty acid oxidation feeds electrons into CII. In reality, dehydrogenases of fatty acid oxidation channel electrons to the Q-junction but not through CII. The ambiguities surrounding Complex II in the literature and educational resources call for quality control, to secure scientific standards in current communications of bioenergetics, and ultimately support adequate clinical applications. This review aims to raise awareness of the inherent ambiguity crisis, complementing efforts to address the well-acknowledged issues of credibility and reproducibility.

Probing the role of protein conformational changes in the mechanism of prenylated-FMN-dependent phenazine-1-carboxylic acid decarboxylase.

Phenazine-1-carboxylic acid decarboxylase (PhdA) is a prenylated-FMN-dependent (prFMN) enzyme belonging to the UbiD family of decarboxylases. Many UbiD-like enzymes catalyze (de)carboxylation reactions on aromatic rings and conjugated double bonds and are potentially valuable industrial catalysts. We have investigated the mechanism of PhdA using a slow turnover substrate, 2,3-dimethylquinoxaline-5-carboxylic acid (DQCA). Detailed analysis of the pH dependence and solvent deuterium isotope effects associated with the reaction uncovered unusual kinetic behavior. At low substrate concentrations, a substantial inverse solvent isotope effect (SIE) is observed on Vmax/KM of ∼ 0.5 when reaction rates of DQCA in H2O and D2O are compared. Under the same conditions, a normal SIE of 4.15 is measured by internal competition for proton transfer to the product. These apparently contradictory results indicate that the SIE values report on different steps in the mechanism. A proton inventory analysis of the reaction under Vmax/KM and Vmax conditions points to a "medium effect" as the source of the inverse SIE. Molecular dynamics simulations of the effect of D2O on PhdA structure support that D2O reduces the conformational lability of the enzyme and results in a more compact structure, akin to the active, "closed" conformer observed in crystal structures of some UbiD-like enzymes. Consistent with the simulations, PhdA was found to be more stable in D2O and to bind DQCA more tightly, leading to the observed rate enhancement under Vmax/KM conditions.

The LOV-domain blue-light receptor LreA of the fungus Alternaria alternata binds predominantly FAD as chromophore and acts as a light and temperature sensor.

Light and temperature sensing are important features of many organisms. Light may provide energy but may also be used by non-photosynthetic organisms for orientation in the environment. Recent evidence suggests that plant and fungal phytochrome and plant phototropin serve dual functions as light and temperature sensors. Here we characterized the fungal LOV-domain blue-light receptor LreA of Alternaria alternata and show that it predominantly contains FAD as chromophore. Blue-light illumination induced ROS production followed by protein agglomeration in vitro. In vivo ROS may control LreA activity. LreA acts as a blue-light photoreceptor but also triggers temperature-shift-induced gene expression. Both responses required the conserved amino acid cysteine 421. We therefore propose that temperature mimics the photoresponse, which could be the ancient function of the chromoprotein. Temperature-dependent gene expression control with LreA was distinct from the response with phytochrome suggesting fine-tuned, photoreceptor-specific gene regulation.

Coupling and regulation mechanisms of the flavin-dependent halogenase PyrH observed by infrared difference spectroscopy.

Flavin-dependent halogenases are central enzymes in the production of halogenated secondary metabolites in various organisms and they constitute highly promising biocatalysts for regioselective halogenation. The mechanism of these monooxygenases includes formation of hypohalous acid from a reaction of fully reduced flavin with oxygen and halide. The hypohalous acid then diffuses via a tunnel to the substrate-binding site for halogenation of tryptophan and other substrates. Oxidized flavin needs to be reduced for regeneration of the enzyme, which can be performed in vitro by a photoreduction with blue light. Here, we employed this photoreduction to study characteristic structural changes associated with the transition from oxidized to fully reduced flavin in PyrH from Streptomyces rugosporus as a model for tryptophan-5-halogenases. The effect of the presence of bromide and chloride or the absence of any halides on the UV-vis spectrum of the enzyme demonstrated a halide-dependent structure of the flavin-binding pocket. Light-induced FTIR difference spectroscopy was applied and the signals assigned by selective isotope labeling of the protein moiety. The identified structural changes in α-helix and ß-sheet elements were strongly dependent on the presence of bromide, chloride, the substrate tryptophan, and the product 5-chloro-tryptophan, respectively. We identified a clear allosteric coupling in solution at ambient conditions between cofactor-binding site and substrate-binding site that is active in both directions, despite their separation by a tunnel. We suggest that this coupling constitutes a fine-tuned mechanism for the promotion of the enzymatic reaction of flavin-dependent halogenases in dependence of halide and substrate availability.

An active site mutation induces oxygen reactivity in D-arginine dehydrogenase: A case of superoxide diverting protons.

Enzymes are potent catalysts that increase biochemical reaction rates by several orders of magnitude. Flavoproteins are a class of enzymes whose classification relies on their ability to react with molecular oxygen (O2) during catalysis using ionizable active site residues. Pseudomonas aeruginosa D-arginine dehydrogenase (PaDADH) is a flavoprotein that oxidizes D-arginine for P. aeruginosa survival and biofilm formation. The crystal structure of PaDADH reveals the interaction of the glutamate 246 (E246) side chain with the substrate and at least three other active site residues, establishing a hydrogen bond network in the active site. Additionally, E246 likely ionizes to facilitate substrate binding during PaDADH catalysis. This study aimed to investigate how replacing the E246 residue with leucine affects PaDADH catalysis and its ability to react with O2 using steady-state kinetics coupled with pH profile studies. The data reveal a gain of O2 reactivity in the E246L variant, resulting in a reduced flavin semiquinone species and superoxide (O2•-) during substrate oxidation. The O2•- reacts with active site protons, resulting in an observed nonstoichiometric slope of 1.5 in the enzyme's log (kcat/Km) pH profile with D-arginine. Adding superoxide dismutase results in an observed correction of the slope to 1.0. This study demonstrates how O2•- can alter the slopes of limbs in the pH profiles of flavin-dependent enzymes and serves as a model for correcting nonstoichiometric slopes in elucidating reaction mechanisms of flavoproteins.

Rapid reaction studies on the chemistry of flavin oxidation in urocanate reductase.

Urocanate reductase (UrdA) is a bacterial flavin-dependent enzyme that reduces urocanate to imidazole propionate, enabling bacteria to use urocanate as an alternative respiratory electron acceptor. Elevated serum levels of imidazole propionate are associated with the development of type 2 diabetes, and, since UrdA is only present in humans in gut bacteria, this enzyme has emerged as a significant factor linking the health of the gut microbiome and insulin resistance. Here, we investigated the chemistry of flavin oxidation by urocanate in the isolated FAD domain of UrdA (UrdA') using anaerobic stopped-flow experiments. This analysis unveiled the presence of a charge-transfer complex between reduced FAD and urocanate that forms within the dead time of the stopped-flow instrument (∼1 ms), with flavin oxidation subsequently occurring with a rate constant of ∼60 s-1. The pH dependence of the reaction and analysis of an Arg411Ala mutant of UrdA' are consistent with Arg411 playing a crucial role in catalysis by serving as the active site acid that protonates urocanate during hydride transfer from reduced FAD. Mutational analysis of urocanate-binding residues suggests that the twisted conformation of urocanate imposed by the active site of UrdA' facilitates urocanate reduction. Overall, this study provides valuable insight into the mechanism of urocanate reduction by UrdA.

Structures and Electron Transport Paths in the Four Families of Flavin-Based Electron Bifurcation Enzymes.

Oxidoreductases facilitating electron transfer between molecules are pivotal in metabolic pathways. Flavin-based electron bifurcation (FBEB), a recently discovered energy coupling mechanism in oxidoreductases, enables the reversible division of electron pairs into two acceptors, bridging exergonic and otherwise unfeasible endergonic reactions. This chapter explores the four distinct FBEB complex families and highlights a decade of structural insights into FBEB complexes. In this chapter, we discuss the architecture, electron transfer routes, and conformational changes across all FBEB families, revealing the structural foundation that facilitate these remarkable functions.

Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging.

Flavin-binding fluorescent proteins are promising genetically encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide, and flavin adenine dinucleotide) are notoriously similar even between different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of 22 finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans. We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512 nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging of bacteria, as well as two-color imaging of mammalian cells (HEK293T), using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of flavin-binding fluorescent proteins in fluorescence microscopy.

A biosynthetic aspartate N-hydroxylase performs successive oxidations by holding intermediates at a site away from the catalytic center.

Nitrosuccinate is a biosynthetic building block in many microbial pathways. The metabolite is produced by dedicated L-aspartate hydroxylases that use NADPH and molecular oxygen as co-substrates. Here, we investigate the mechanism underlying the unusual ability of these enzymes to perform successive rounds of oxidative modifications. The crystal structure of Streptomyces sp. V2 L-aspartate N-hydroxylase outlines a characteristic helical domain wedged between two dinucleotide-binding domains. Together with NADPH and FAD, a cluster of conserved arginine residues forms the catalytic core at the domain interface. Aspartate is found to bind in an entry chamber that is close to but not in direct contact with the flavin. It is recognized by an extensive H-bond network that explains the enzyme's strict substrate-selectivity. A mutant designed to create steric and electrostatic hindrance to substrate binding disables hydroxylation without perturbing the NADPH oxidase side-activity. Critically, the distance between the FAD and the substrate is far too long to afford N-hydroxylation by the C4a-hydroperoxyflavin intermediate whose formation is confirmed by our work. We conclude that the enzyme functions through a catch-and-release mechanism. L-aspartate slides into the catalytic center only when the hydroxylating apparatus is formed. It is then re-captured by the entry chamber where it waits for the next round of hydroxylation. By iterating these steps, the enzyme minimizes the leakage of incompletely oxygenated products and ensures that the reaction carries on until nitrosuccinate is formed. This unstable product can then be engaged by a successive biosynthetic enzyme or undergoes spontaneous decarboxylation to produce 3-nitropropionate, a mycotoxin.

A new member of the flavodoxin superfamily from Fusobacterium nucleatum that functions in heme trafficking and reduction of anaerobilin.

Fusobacterium nucleatum is an opportunistic oral pathogen that is associated with various cancers. To fulfill its essential need for iron, this anaerobe will express heme uptake machinery encoded at a single genetic locus. The heme uptake operon includes HmuW, a class C radical SAM-dependent methyltransferase that degrades heme anaerobically to release Fe2+ and a linear tetrapyrrole called anaerobilin. The last gene in the operon, hmuF encodes a member of the flavodoxin superfamily of proteins. We discovered that HmuF and a paralog, FldH, bind tightly to both FMN and heme. The structure of Fe3+-heme-bound FldH (1.6 Å resolution) reveals a helical cap domain appended to the ⍺/ß core of the flavodoxin fold. The cap creates a hydrophobic binding cleft that positions the heme planar to the si-face of the FMN isoalloxazine ring. The ferric heme iron is hexacoordinated to His134 and a solvent molecule. In contrast to flavodoxins, FldH and HmuF do not stabilize the FMN semiquinone but instead cycle between the FMN oxidized and hydroquinone states. We show that heme-loaded HmuF and heme-loaded FldH traffic heme to HmuW for degradation of the protoporphyrin ring. Both FldH and HmuF then catalyze multiple reductions of anaerobilin through hydride transfer from the FMN hydroquinone. The latter activity eliminates the aromaticity of anaerobilin and the electrophilic methylene group that was installed through HmuW turnover. Hence, HmuF provides a protected path for anaerobic heme catabolism, offering F. nucleatum a competitive advantage in the colonization of anoxic sites of the human body.

High sensitivity and low-cost flavin luciferase (FLUXVc)-based reporter gene for mammalian cell expression.

Luciferase-based gene reporters generating bioluminescence signals are important tools for biomedical research. Amongst the luciferases, flavin-dependent enzymes use the most economical chemicals. However, their applications in mammalian cells are limited due to their low signals compared to other systems. Here, we constructed Flavin Luciferase from Vibrio campbellii (Vc) for Mammalian Cell Expression (FLUXVc) by engineering luciferase from V. campbellii (the most thermostable bacterial luciferase reported to date) and optimizing its expression and reporter assays in mammalian cells which can improve the bioluminescence light output by >400-fold as compared to the nonengineered version. We found that the FLUXVc reporter gene can be overexpressed in various cell lines and showed outstanding signal-to-background in HepG2 cells, significantly higher than that of firefly luciferase (Fluc). The combined use of FLUXVc/Fluc as target/control vectors gave the most stable signals, better than the standard set of Fluc(target)/Rluc(control). We also demonstrated that FLUXVc can be used for testing inhibitors of the NF-κB signaling pathway. Collectively, our results provide an optimized method for using the more economical flavin-dependent luciferase in mammalian cells.

Deciphering the biosynthesis of a novel lipid in Mycobacterium tuberculosis expands the known roles of the nitroreductase superfamily.

Mycobacterium tuberculosis's (Mtb) success as a pathogen is due in part to its sophisticated lipid metabolic programs, both catabolic and biosynthetic. Several of Mtb lipids have specific roles in pathogenesis, but the identity and roles of many are unknown. Here, we demonstrated that the tyz gene cluster in Mtb, previously implicated in resistance to oxidative stress and survival in macrophages, encodes the biosynthesis of acyl-oxazolones. Heterologous expression of tyzA (Rv2336), tyzB (Rv2338c) and tyzC (Rv2337c) resulted in the biosynthesis of C12:0-tyrazolone as the predominant compound, and the C12:0-tyrazolone was identified in Mtb lipid extracts. TyzA catalyzed the N-acylation of l-amino acids, with highest specificity for l-Tyr and l-Phe and lauroyl-CoA (kcat/KM = 5.9 ± 0.8 × 103 M-1s-1). In cell extracts, TyzC, a flavin-dependent oxidase (FDO) of the nitroreductase (NTR) superfamily, catalyzed the O2-dependent desaturation of the N-acyl-L-Tyr produced by TyzA, while TyzB, a ThiF homolog, catalyzed its ATP-dependent cyclization. The substrate preference of TyzB and TyzC appear to determine the identity of the acyl-oxazolone. Phylogenetic analyses revealed that the NTR superfamily includes a large number of broadly distributed FDOs, including five in Mtb that likely catalyze the desaturation of lipid species. Finally, TCA1, a molecule with activity against drug-resistant and persistent tuberculosis, failed to inhibit the cyclization activity of TyzB, the proposed secondary target of TCA1. Overall, this study identifies a novel class of Mtb lipids, clarifies the role of a potential drug target, and expands our understanding of the NTR superfamily.

The crystal structure of Vibrio cholerae (6-4) photolyase reveals interactions with cofactors and a DNA-binding region.

Photolyases (PLs) reverse UV-induced DNA damage using blue light as an energy source. Of these PLs, (6-4) PLs repair (6-4)-lesioned photoproducts. We recently identified a gene from Vibrio cholerae (Vc) encoding a (6-4) PL, but structural characterization is needed to elucidate specific interactions with the chromophore cofactors. Here, we determined the crystal structure of Vc (6-4) PL at 2.5 Å resolution. Our high-resolution structure revealed that the two well-known cofactors, flavin adenine dinucleotide and the photoantenna 6,7-dimethyl 8-ribityl-lumazin (DMRL), stably interact with an α-helical and an α/ß domain, respectively. Additionally, the structure has a third cofactor with distinct electron clouds corresponding to a [4Fe-4S] cluster. Moreover, we identified that Asp106 makes a hydrogen bond with water and DMRL, which indicates further stabilization of the photoantenna DMRL within Vc (6-4) PL. Further analysis of the Vc (6-4) PL structure revealed a possible region responsible for DNA binding. The region located between residues 478 to 484 may bind the lesioned DNA, with Arg483 potentially forming a salt bridge with DNA to stabilize further the interaction of Vc (6-4) PL with its substrate. Our comparative analysis revealed that the DNA lesion could not bind to the Vc (6-4) PL in a similar fashion to the Drosophila melanogaster (Dm, (6-4)) PL without a significant conformational change of the protein. The 23rd helix of the bacterial (6-4) PLs seems to have remarkable plasticity, and conformational changes facilitate DNA binding. In conclusion, our structure provides further insight into DNA repair by a (6-4) PL containing three cofactors.