Intramolecular Coupling and Nucleobase Transfer – How Cytochrome P450 Enzymes GymBx Establish Their Chemoselectivity

Cyclodipeptides (CDPs) build an important class of secondary metabolites with a plethora of pharmacological impact such as antibiotic, cytotoxic, antifungal properties or even as anti‐Alzheimer drug candidates. The core structure of 2,5‐diketopiperazines, a subclass of CDPs, is composed of two ‘head‐to‐tail’ connected α‐amino acids. Their chemical diversity is broadened by tailoring enzymes as members of their biosynthetic gene clusters, such as CDP‐dimerizing P450s. Recently, a new class of P450 enzymes, termed GymBx, was identified in the biosynthetic pathway of guatyromycines. These enzymes catalyse both an intramolecular C−C bond formation within cyclo‐l‐Tyr‐l‐Tyr (cYY) and a nucleobase transfer reaction, which is unique for CDP‐modifying P450 enzymes. In this study, we report the structures of two members, GymB1 and GymB5, in their unbound states, as well as GymB5 in complex with hypoxanthine and cYY. Structural data of the ternary complex shed light into CDP binding and nucleobase transfer reaction, identified key residues for substrate recognition and modified the enzymatic bifunctionality. By mutagenesis we successfully established a GymB5 variant that strictly switched the chemoselectivity of the reaction towards an intermolecular coupling enzyme. This data set a solid basis for future protein engineering of P450 enzymes to synthesize new CDP‐nucleobase adducts.


Introduction
Natural products obtained from the secondary metabolism of bacteria, fungi or plants have gained growing interest for biological and pharmacological research, due to their vast variety of medically relevant properties, such as anti-oxidative, antifungal, antibacterial, cytotoxic, and anti-Alzheimer impact. [1,2,3,4,5]Especially, cyclodipeptides (CDPs) with 2,5-diketopiperazine (DKP) building blocks have gained importance as they are highly cell permeable due to their small size and lipophilic feature, making them interesting targets for drug delivery and development.In nature, cyclodipeptides containing a DKP core are formed by condensation of two α-amino acids, either catalyzed by nonribosomal peptide synthetases (NRPSs) or cyclodipeptide synthases (CDPSs). [6,7,8]Very recently, another biosynthetic route for the synthesis of argininecontaining cyclic dipeptides (RCDPSs) was identified in a fungal host. [9]The resulting scaffolds can then further be modified by so-called tailoring enzymes, such as cytochrome P450 enzymes (P450s), cyclodipeptide oxidases, 2-oxoglutarate and Fe II -dependent oxygenases, methyltransferases, prenyltransferases, and terpene cyclases, to yield a plethora of various secondary metabolites. [10,11,12,13,14]450s are ubiquitously found in all domains of life.In eukaryotes, these enzymes fulfil a plethora of functions with a key role in the metabolism of steroids, vitamins, carcinogens, drugs and other xenobiotics. [15,16]In bacteria and fungi, this enzyme superfamily is often associated to biosynthetic gene clusters of secondary metabolites.Although structurally highly conserved, these enzymes have diverse catalytic functions, such as hydroxylations, epoxidations or coupling reactions. [17]Their high degree of regio-and stereospecificity of the catalyzed reactions render them valuable tools for bioenzymatic synthesis. [18]P450s within the CDPS-containing gene clusters came into the focus of research, as they showed a remarkable diversity of their catalyzed reactions (Figure 1), which further broaden the chemical diversity of natural compounds. [10,12]urther protein engineering of CDP-modifying P450s showed their potential for future chemical diversity by chemoenzymatic synthesis. [19]Currently, a very limited number of CDP-modifying P450s are structurally characterized [20,21,22,23,24] and their mechanism of catalysis is under investigation. [25]Recently, we have identified a new class of P450s that are able to catalyze the transfer of a nucleobase onto a cyclodipeptide, a unique reaction that was not described for P450s so far.This new enzymatic reaction yields to the formation of new classes of secondary metabolites termed guanitrypmycines, guatrypmethines and guatyromycines. [26,27,28,29]In the biosynthetic pathway for the latter, the homologous CDPSs GymA 1-6 were found to assemble the cyclodipeptide scaffolds cyclo-l-Tyr-l-Tyr (cYY) and cyclo-l-Tyr-l-Phe (cYF) in six different Streptomyces strains.The associated P450 enzymes, termed GymB 1-6 , then catalyze the transfer of either nucleobase guanine or hypoxanthine onto the cyclodipeptide or the formation of mycocyclosin by intra-molecular CÀ C coupling (Figure 1), an enzymatic reaction that was also reported for CYP121 from Mycobacterium tuberculosis. [20]The bifunctionality of these enzymes to either perform an intramolecular CDP coupling or a nucleobase transfer is particularly interesting but lack structural data to elucidate how either of both reactions are achieved.An additional promiscuity arises from the observation that the nucleobase transfer yields to the formation of either CÀ O bonds as well as modifying the carbon skeleton by the formation of a CÀ C bond from guanine or hypoxanthine to cYY (see Figure 1).The catalytic promiscuity of the GymB x enzymes render them as valuable chemical tools to broaden the chemical space of CDPs.Although structures of P450 enzymes involved in the cyclodipeptide dimerization were solved recently, [19,22,23] giving insight into possible binding patterns for these scaffolds, to the best of our knowledge, structural data on coupling reactions with guanine or hypoxanthine of GymB x enzymes that would explain how these enzymes recognize their substrates and how they control their chemoselectivity remains to be determined.
In this study, we set out to structurally explore the unusual GymB x reaction and present structures of CDP-nucleobase forming P450s GymB 1 and GymB 5 , in their unbound state, as well as bound to both natural substrates cYY and hypoxanthine for GymB 5 .To the best of our knowledge, these are the first structures of a nucleobase transferring P450 enzyme.The structural data allowed us to identify how this enzyme class recognizes their natural substrates and which key residues control the bifunctionality.

Structure Determination of GymB x Enzymes
Recently, we identified and biochemically characterized a new type of bifunctional CDP-modifying cytochrome P450 enzymes from CDPS-containing biosynthetic gene clusters in six different Streptomyces species. [26]These enzymes possess a high sequence identity of 66-79 % (Table S1) and are able to transfer a nucleobase (either guanine or hypoxanthine) onto a CDP or to catalyze its intramolecular coupling.Thus, we selected two homologous enzymes of this family based on their expression level in E. coli, i. e. GymB 1 from Streptomyces flavidovirens and GymB 5 from Streptomyces katrae.GymB 5 shares a sequence identity of 68 % (Table S1) with GymB1 and both catalyze a similar guanine or hypoxanthine transfer to CDPs such as cYY or cYF.Codon-optimized constructs of the two genes were used to recombinantly produce the enzymes in E. coli.The purified proteins were subjected to crystallization experiments.The resulting crystals of the apo-enzymes yielded structures of unliganded GymB 1 (termed apoGymB 1 ) and GymB 5 (termed apoGymB 5 ) to high-resolution of 1.96 Å and 1.90 Å, respectively (Table 1).The ternary complex of GymB 5 with hypoxanthine and cYY was formed by co-crystallization experiments.We were able to boost the occupancy of cYY in the active site by soaking precharged crystals obtained from co-crystallization trials with high concentrations of cYY and hypoxanthine.The resulting structure GymB 5 /cYYhypo was established at 1.80 Å resolution and unequivocally showed the presence of both substrates in one of both protomers of the asymmetric unit (ASU).All structures were refined to excellent quality standards (Table 1), validated, and deposited to the protein data bank.Noteworthy, the second protomer harbored two hypoxanthine molecules in the active site, lacking the cYY substrate.The cYY could not be found in the second chain of GymB 5 /cYYhypo probably because helix B2' that is involved in CDP binding could not undergo the necessary structural rearrangement due to crystal contacts keeping GymB 5 in its opened state.Therefore, cYY could not be trapped in this protomer probably due to its free diffusion.

The Overall Structures of GymB x Resemble a Classical Cytochrome P450 Fold
Both GymB 1 and GymB 5 form monomers and dimers in solution as confirmed by analytical SEC.Bioinformatics predictions based on our structural data calculated with EPPIC [30] suggested a biological dimer (Figure 2A) with high probability formed by a conserved crystal contact found in both crystal packings of GymB 1 and GymB 5 (Tables S2).
A similar crystal contact was also observed in CYP121 from Mycobacterium tuberculosis characterized as monomeric). [20]Both protomers are linked via a two-fold rotation symmetry that is either non-crystallographic as for monoclinic crystals of GymB 5 , or part of the crystallographic symmetry for the trigonal packing of apoGymB 1 crystals.The dimer interface buries a solvent accessible area (BSA) of 1385 Å 2 and 1217 Å 2 for GymB 1 and GymB 5 , respectively.We observed a concentration dependent distribution of the dimeric species by analytical SEC (Figure S2) which would suggest that both GymB 1 and GymB 5 form monomers under physiological concentration in their native environment.
As expected by its high degree of sequence similarity, GymB 5 and GymB 1 are structurally closely related enzymes possessing a C α -RMSD of 0.85 Å when superposing both protein chains.Both proteins fold into a typical cytochrome P450 prismlike fold (Figure 2), [31] consisting of 15 α-helices and two antiparallel β-sheets (Figures 2A and 2B).A search for structural homologues using the DALI [32] server revealed that GymB 5 is structurally closely related to CYP121 from Mycobacterium tuberculosis, a cytochrome P450 enzyme that utilizes the same substrate (cYY) to form mycocyclosin via an intramolecular cyclization reaction (Table S3). [20]Both sequence identity of this enzyme of 57 % (Table S4) and the C α -RMSD of 1.1 Å suggest a virtual identical fold of the homologues.Further structural homologues include cytochrome P450 enzymes from different Streptomyces strains, such as SvgP from Streptomyces griseoviridis (C α -RMSD 2.1 Å) (pdb-code: 4MM0), TleB from Streptomyces blastomyceticus (C α -RMSD 2.3 Å, pdb code 6J83) [21] or NzeB from Streptomyces sp.NRRL F-5053 (C α -RMSD 2.3 Å, pdb code 6XAI). [23]apping of the C α -RMSD values onto GymB 5 (Figure S3) showed that the core fold including helices that surround the heme group (I, L, K) as well as helices D and E are structurally very well conserved.Main differences occur in the loop regions as well as in the conformation of helices B2 and B2'.This region of cytochrome P450 enzymes is known to be involved in substrate binding [33] and structural flexibility is in accordance with substrate binding capabilities to catalyze various chemical reactions.

Substrate Binding Induces Conformational Changes Around the Active Site
The active site of GymB 5 is formed by the central heme molecule sitting below the long helix I, the signature helix of cytochrome P450 enzymes running across the entire protein molecule and containing a conserved serine or threonine residue at its center that is crucial for catalysis. [34]Here, residues T226, G229, A230, I233 and S234 form one wall of the reaction chamber for CDP-nucleobase coupling.Furthermore, residues of helices B2 (L61) and B2' (T76, I77, V81, V82, N84), helix α8 (A166, F167), helix α9 (W181), the loop α13β3 (residues I277, D279, G280 and L281), a C-terminal loop region (M382, K383) shape the cavity of the active site (Figure 3A).Upon substrate binding, GymB 5 undergoes a substantial induced fit (Figures 3A, 3B,S4 and S5).A comparison of the substrate-bound versus the substrate-free state of GymB 5 revealed differences in both structures.In the open state the active site cavity is enlarged by the conformation of helices B2/ B2' and helix B. By substrate binding, a large portion of helices B2/B2' (residues 76 to 93), moves into the previously open cavity.This closes the active site and forms the reaction chamber that shields the catalytic reaction from the solvent.
The observed induced fit is crucial for substrate recognition as it contributes to shape complementarity of cYY and introduces hydrogen bonds to the DKP ring system which are backbone atoms of T76, V82 and M85 and a direct hydrogen bond to N84 (Figure 3A and 3B).These interactions would be impossible in the open state of the enzyme.The overall C α -RMSD between residues 76 and 93 compared to both states is 3.1 Å, while overall the C α -RMSD for the remaining residues showed a virtual identical conformation (C α -RMSD 0.2 Å).Open and closed conformations have been reported for some of the structural homologues of GymB 5 , such as OleP [35] (pbd code: 5MNS) and CYP105P1 (pdb code: 3ABA) . [36]However, structural rearrangements take place between the FG helices and BC loop of the enzymes, displaced from the induced fit that was observed for GymB 5 .It has been proposed that the open conformation allows for entry of the larger substrates, while closing of the active site positions enables both substrates in close proximity to the heme group for catalysis. [35]We conclude that the induced fit serves the same purpose and hypothesize that the enzyme consequently evolved a different opening and closing mechanism to allow for large substrate entry and subsequent placement suitable for catalysis.Noteworthy, a comparable induced fit is not observed in the closely related homologue CYP121 from Mycobacterium tuberculosis in the reported structures, but it remains unclear if this is an effect of crystal packing.
Both substrates (cYY and hypoxanthine) were unambiguously placed in the active site of GymB 5 based on an unbiased simulated annealing omit map (Figures 3A, 3B).The substrates tightly fit into the active site of GymB 5 with mainly hydrophobic interactions.The active site can be subdivided into a cYY site and a guanine/hypoxanthine site.Only a limited set of direct hydrogen bonds recognizes both substrates.On the hypoxan- thine site, these are K383 and D279 forming hydrogen bonds to the carbonyl group and amide nitrogen of hypoxanthine, respectively.In addition, a water-mediated interaction of the carboxylic entity of heme towards atom N7 of hypoxanthine contributes to substrate recognition.The remaining interactions are characterized by tight hydrophobic packing to residues M382, I277 and L281.Although we were not able to establish a guanine complex with GymB 5 , it is noteworthy that guanine could adopt the same binding orientation without substantial steric repulsion of the additional amino moiety with the protein skeleton.
cYY adopts a U-shaped conformation with respect to the piperazine ring system.On the cYY binding site a single direct hydrogen bond to the carbonyl atom of the DKP ring system is formed by N84, but three water-mediated hydrogen interactions to the backbone atoms of V82, M85, A230 and T76 and to the side chain of S230 were observed (Figure 3).The distal tyrosine residue of cYY is tightly packed by hydrophobic van der Waals contacts of I77, F167 and W181.The hydroxy group of the proximal tyrosine of cYY is positioned on top of the iron heme to which it is coordinated via a water molecule.The reaction center is built by the atoms C8 of hypoxanthine and the hydroxy group and the atom C6 of the proximal tyrosine (ortho position with respect to the hydroxy group).An analysis of the intermolecular distances between both substrates revealed that the oxygen atom of the proximal tyrosine is in close contact with C8 of hypoxanthine possessing an OH to C8 atom distance of 3.3 Å.The closest distance to establish a covalent intermolecular CÀ C bond without losing the aromaticity of the tyrosine ring system of cYY is found for atom C6 of cYY and C8 of hypoxanthine positioned 3.6 Å away.The formation of guatyromycine B is less favoured due to a larger distance of C6 of cYY to C3 of hypoxanthine of 5.7 Å which seem to include a resonance stabilized radical of hypoxanthine.In summary, the active site geometry of both substrates is in perfect agreement with the catalyzed reaction forming both guatyromycines but with a high specificity to establish the CÀ O bond as observed for guatyromycine A. Noteworthy, all GymB x enzymes characterized so far share identical amino acid residues involved in substrate binding (Figure S6) which let us to conclude that the data provided here should also be directly applicable to these members and only subtle changes in the kinetics might be observed.

Comparison to Other CDP-Modifying P450s
We first compared the active sites of the closest structural homologs.Although the overall fold of CDP-dimerizing P450s and GymB5 is structurally well conserved, the active sites of these enzymes do not superpose well and possess various differences (Figure 4A). Next, we focused on CDPutilizing P450 enzymes which have been characterized to dimerize CDPs.Members of these enzymes are currently in the focus of research and have been recently biochemically characterized and some of them have been structurally determined in complex with their substrate (NzeB, AspB, NasF5053). [22,37,38,23]Although the cYY binding site of GymB 5 that is occupied with cyclo-(l-Trp-l-Pro) (cWP, also known as brevianamide F) in these proteins, adopts a comparable conformation for both CDPs, structural differences mainly caused by the amino acid pair N84/T226 in that binding site result in a lift up of cYY by approximately 2 Å away from the heme group (Figure 4A).In the dimerizing CDP enzymes, the amino acid pair is substituted by a pair of non-polar side chains.N84 is substituted by a small amino acid, mainly alanine or glycine, whereas the T226 is replaced by a large hydrophobic residue, mainly leucine or methionine.As a consequence, the direct hydrogen bond formed by N84 to the DKP carbonyl could not be observed.As the CDP-binding site is very comparable to CDP-dimerizing enzymes (NzeB, AspB, NasF5053), we focused to identify key residues that control the specificity of the chemical reaction to drive either an intermolecular coupling/dimerization of CDPs versus a nucleobase transfer reaction.Our data clearly suggests that the guanine/ hypoxanthine binding site (GHBS) of GymB 5 and not the CDPbinding site is in focus for further protein engineering.This conclusion is also supported by recent data of the CDP dimerizing enzyme NasF 5053 .The authors demonstrated that protein engineering of this enzyme yields variants that are capable to produce 12 self-dimerizing and at least 81 crossdimerized heterodimeric tryptophan-containing diketopiperazines (HTDKPs) just by exchange of three crucial amino acids (F387, F388 and E73). [19]By structure-based sequence comparison, these residues correspond to M382, K383 and Y73 in GymB 5 , respectively.Noteworthy, two of the three positions are located at the guanine binding site (F387, F388).When comparing the guanine/hypoxanthine binding site (GHBS), it is remarkable that the aromatic ring location of the CDP in dimerizing P450s is comparable to the hypoxanthine position, but the guanine ring adopts an almost 180°rotated conformation (Figure 4A, Figure S7).The binding of a second CDP in the GHBS is directly sterically hampered by three residues D279, I277 and Y73 of which the latter side chain corresponds to E73 in NasF 5053 , whereas D279 and I277 are replaced by small hydrophobic residues (mainly A or G) in dimerizing enzymes.Another important variation of GymB 5 versus dimerizing P450s seems to be in position P282 of GymB 5 , which is replaced by a lysine residue in dimerizing P450s.Noteworthy, the conserved lysine residue in dimerizing P450s is involved in bridging the carbonyl moieties of both DKP carbonyl atoms.In summary, our structure somparison shows crucial differences in position N84, T226, D279, I277, Y73 and P282 that would exclude the intermolecular coupling of two CDPs.Thereby, we set solid basis to distinguish dimerizing P450 from the nucleobase transferring P450s of the GymB 5 family.

Controlling the Nucleobase Transfer
Finally, we set out to explore differences of GymB 5 towards CYP121 that utilizes the same substrate cYY to produce mycocyclosin via an intramolecular coupling reaction.Therefore, it is expected that the active sites share common features in the recognition of this substrate and differences identified to dimerizing P450 are also found for CYP121.Indeed, concerning cYY binding only subtle changes are observed and the replacement of only two amino acids, namely L61 M, I77 V would transform the active site from GymB 5 to CYP121.Similar interactions are observed for cYY recognition and the shape and orientation of cYY are virtually identical (Figure 4B, Figure S7).On the GHBS the changes are more pronounced to compensate for the additional space necessary to accommodate a guanine or hypoxanthine molecule.Here, point mutations M382Q, K383R and most importantly I277F could reshape the active site from GymB 5 to CYP121.Amino acid I277 forms tight packing towards hypoxanthine in GymB 5 and its exchange to a bulky and rigid phenyl side chain would directly sterically interfere with its accommodation in the active site, whereas the remaining two substitutions are either functional conserved (K383R) or do not hamper hypoxanthine or guanine binding.Wildtype GymB5 showed a clear prevalence in the formation of guatyromycine A over mycocyclosin by a ratio of 6.7 : 1. Mutation of F280 in CYP121 to the smaller isoleucine might therefore allow CYP121 to undergo a nucleobase transfer reaction.
Based on our structural data, we speculated that position I277 establishes the key feature to switch from an intermolecular substitution towards an intramolecular coupling reaction and concluded that the chemoselectivity of the reaction is controlled at this position.Therefore, we set out to explore the chemoselectivity switch by site-directed mutagenesis.Structural comparison suggested the most obvious candidate GymB 5 _ I277F.Although LC-MS data clearly indicated a specificity switch of GymB 5 _I277F towards mycocyclosin formation (Figure 5, S8), the turnover rates for this candidate were limited and the obtained chemoselectivity did not fulfil our quality criteria although clearly indicating a prevalence for intramolecular coupling.Therefore, we extended our investigation by the introduction of bulky and positively charged residues in GymB 5 _I277K and GymB 5 _I277R with the intention to still sterically block the GHBS but also to allow for direct hydrogen  bonding towards the hydroxy group of cYY which should enhance the intermolecular coupling reaction.
Variant GymB 5 _I277K substantially enhanced the enzymatic efficiency of the enzyme (Table 2) and clearly showed a prevalence for mycocyclosin formation (Figure 5) but still retained capable for the wildtype reaction, properly due to intrinsic flexibility in the lysine side chain or because lysine is too short to reach out the cYY binding site.It is noteworthy that variant GymB 5 _I277K also produces a not yet characterized product.A structural characterization of this substance is currently hampered by the amount of material.
Encouraged by the results of GymB5_I227K, we generated a mutant GymB 5 _I277R.This mutant did not show any wild-type reaction at all, but exclusively results in the formation of mycocyclosin (Figure 5) with an excellent catalytic efficiency of approximately 8400 s À 1 M À 1 .Although we could not provide structural data for these variants, the kinetic data suggest that the introduction of the most protruding side chain arginine sufficiently enlarge the enzyme to reach out for cYY interaction.In summary, we identified the enzymatic key residue that controls the chemoselectivity of intramolecular coupling versus nucleobase transfer reaction in GymB 5 and established a GymB 5 variant that strictly switches the catalysis towards intramolecular coupling but still shows decent catalytic efficiency.

Conclusions
In this work, we report crystal structures of a new class of CDP modifying P450 enzymes GymB x , which is capable to perform a nucleobase transfer onto the CDP cYY.Moreover, these enzymes are catalytically promiscuous and also allow for an intramolecular coupling reaction to produce mycocyclosin, the product of CYP121 from Mycobacterium tuberculosis.Analysis of the structural data of the ternary complex of GymB 5 with cYY and hypoxanthine allowed us to identify key residues for catalysis including residue I277 as a key player in controlling the catalytic promiscuity of the enzyme.Mutation at this position to a bulky phenylalanine (GymB 5 _I277F) residue altered the observed product profile and substantially switched the prevalence of the catalyst towards the intramolecular coupling reaction although with reduced catalytic efficiency.When introducing a lysine residue at this position to establish variant GymB 5 _I277K, we did not only switch the enzymatic specificity towards intramolecular coupling but also enhanced the catalytic turnover of this variant.
In a final attempt, we produced GymB 5 _I277R.This variant is specific fully converts the functionality of the enzyme from a guanylating / hypoxanthinylating enzyme to a CYP121 variant exclusively catalyzing the intramolecular coupling reaction.
Our structural and biochemical data contribute to the general understanding of how CDP-modyfing P450 enzymes establish their substrate and chemoselectivity and structurebased sequence comparision should allow researchers to distinguish between both P450 families.This work substantially contributes to our understanding of the molecular basis of catalysis and will support further protein engineering studies to unleash the great potential by the production of new and adjustable CDPs with pharmacological impact.Future studies might result in the generation of chimeric CPD modifying P450s to enable access to yet uncharacterized DKP compounds.

Figure 1 .
Figure 1.Exemplary representation of the diversity of chemical reactions catalyzed by P450 enzymes attached to CDPS gene clusters.The P450 enzymes are annotated in bold letters.If a protein structure is available, the enzymes are highlighted by gray background.The chemical reaction catalyzed by GymB x enzymes are highlighted by pink background.GymB x proteins are bifunctional catalyzing either intramolecular coupling of cYY or its intermolecular coupling with guanine or hypoxanthine, yielding three possible products.

Figure 2 .
Figure 2. Structure and topology of GymB 5 .A) Crystal structure of GymB 5 including its potential dimeric state.Both protein chains of the enzyme are depicted as cartoon representation, with one protomer highlighted in forest with smudge β-sheets while another one is colored in grey.All ligands and the heme cofactor are represented as sticks and colored in chartreuse, hot pink and cyan, respectively.The helices are labeled according to the typical prism-like fold of cytochrome P450 enzymes.The two-fold non-crystallographic symmetry axis to resemble the dimer is shown as a black line.B) Topology plot of GymB 5 as depicted in A).Residues involved in the binding of the substrates are emphasized by cyan spheres.Residues mutated in this work are shown in orange.The heme group and its coordinating cysteine are drawn schematically in green and yellow, respectively.

Figure 3 .
Figure 3. Substrate Interactions and Active Site Comparison of GymB 5 .A) Side view of active site.The simulated annealing omit maps for both ligands cYY and hypoxanthine are shown at contour level 3s in grey, respectively.Interacting residues are depicted as forest sticks, hydrogen bonds are represented as black dashed lines.The helices performing an induced fit upon substrate binding are represented as sticks and ribbon in the closed state (dark green), whereas the same region of the unliganded structure is superposed as ribbon and colored in lemon.The direction of the induced fit from opened to closed state is indicated by a green arrow.Water molecules are depicted as red spheres.Residues chosen for mutagenesis are represented as orange sticks.Parts of the structure were removed for better visualization of the active site.B) Top view of A).C) Scheme presentation of the active site interactions colored as in A) and B).The orange dashed line illustrates the distances to the reaction centers.On the cYY site the intermediate species might occur either at the hydroxy group or at its ortho position of cYY.Main chain anchor points are depicted as gray circles.Water molecules are represented as burgundy circles.Hydrogen bonds, ionic interactions and Van-der-Waals interactions are shown as black dashed lines.

Figure 4 .
Figure 4. Comparison of the active sites of GymB 5 to CDP dimerizing P450 NzeB and intramolecular coupling enzyme CYP121 by stereo representation.(A) Superposition of GymB 5 to NzeB.Residues of GymB 5 are shown in green whereas residues belonging to NzeB are coloured in grey.The substrate cWP of NzeB and cYY of GymB 5 is shown in grey, respectively.The heme group is shown in light green.Hypoxanthine is shown in cyan.For better readability, side chains of NzeB that adopt a comparable conformation as observed in the active site of GymB 5 are not shown.The labelling is based on the sequence of GymB 5 and includes the corresponding amino acid of NzeB resulting from a structure-based sequence alignment.Position I277 was identified to be crucial for hypoxanthine binding (orange).The dipeptide cWP is bound deeper in the cavity of NzeB compared to cYY in GymB5 as indicated by a black arrow.Most severe clashes of the substrates with proteinaceous side chains are highlighted by transparent spheres in magenta B) Superposition of GymB 5 to intramolecular coupling enzyme CYP121.Colour coding and representation is consistent with A), but grey residues and labelling refers to CYP121.

Table 1 .
Crystallographic Data Collection and Refinement Statistics.
* Hypoxanthine in chain A is annotated as HPA(A), whereas hypoxanthines found in chain B are termed HPA(B).