Diversification of catalytic function in a synthetic family of chimeric cytochrome p450s

We report initial characterization of a synthetic family of more than 3000 cytochrome P450s made by SCHEMA recombination of 3 bacterial CYP102s. A total of 16 heme domains and their holoenzyme fusions with each of the 3 parental reductase domains were tested for activity on 11 different substrates. The results show that the chimeric enzymes have acquired significant functional diversity, including the ability to accept substrates not accepted by the parent enzymes. K-means clustering analysis of the activity data allowed the enzymes to be classified into five distinct groups based on substrate specificity. The substrates can also be grouped such that one can be a "surrogate" for others in the group. Fusion of a functional chimeric heme domain with a parental reductase domain always reconstituted a functional holoenzyme, indicating that key interdomain interactions are conserved upon reductase swapping.     

DNA-mediated assembly of cytochrome P450 BM3 subdomains

Cytochrome P450 BM3 is a versatile enzyme, which holds great promise for applications in biocatalysis and biomedicine. We here report on the generation of a hybrid DNA-protein device based on the two subdomains of BM3, the reductase domain BMR and the porphyrin domain BMP. Both subdomains were fused genetically to the HaloTag protein, a self-labeling enzyme, allowing for the bioconjugation with chloroalkane-modified oligonucleotides. The subdomain-DNA-chimeras could be reassembled by complementary oligonucleotides, thus leading to reconstitution of the monooxygenase activity of BM3 holoenzyme, as demonstrated by conversion of the reporter substrate 12-pNCA. Arrangement of the two chimeras on a switchable DNA scaffold allowed one to control the distance between both subdomains, as indicated by the DNA-dependent activity of the holoenzyme. Furthermore, a switchable chimeric device was constructed, in which monooxygenase activity could be turned off by DNA strand displacement. This study demonstrates that P450 BM3 engineering and strategies of DNA nanotechnology can be merged to open up novel ways for the development of novel screening systems or responsive catalysts with potential applications in drug delivery.     

Two sequence elements of glycosyltransferases involved in urdamycin biosynthesis are responsible for substrate specificity and enzymatic activity.

BACKGROUND: Two deoxysugar glycosyltransferases (GTs), UrdGT1b and UrdGT1c, involved in urdamycin biosynthesis share 91% identical amino acids. However, the two GTs show different specificities for both nucleotide sugar and acceptor substrate. Generally, it is proposed that GTs are two-domain proteins with a nucleotide binding domain and an acceptor substrate site with the catalytic center in an interface cleft between these domains. Our work aimed at finding out the region responsible for determination of substrate specificities of these two urdamycin GTs. RESULTS: A series of 10 chimeric GT genes were constructed consisting of differently sized and positioned portions of urdGT1b and urdGT1c. Gene expression experiments in host strains Streptomyces fradiae Ax and XTC show that nine of 10 chimeric GTs are still functional, with either UrdGT1b- or UrdGT1c-like activity. A 31 amino acid region (aa 52-82) located close to the N-terminus of these enzymes, which differs in 18 residues, was identified to control both sugar donor and acceptor substrate specificity. Only one chimeric gene product of the 10 was not functional. Targeted stepwise alterations of glycine 226 (G226R, G226S, G226SR) were made to reintroduce residues conserved among streptomycete GTs. Alterations G226S and G226R restored a weak activity, whereas G226SR showed an activity comparable with other functional chimeras. CONCLUSIONS: A nucleotide sugar binding motif is present in the C-terminal moiety of UrdGT1b and UrdGT1c from S. fradiae. We could demonstrate that it is an N-terminal section that determines specificity for the nucleotide sugar and also the acceptor substrate. This finding directs the way towards engineering this class of streptomycete enzymes for antibiotic derivatization applications. Amino acids 226 and 227, located outside the putative substrate binding site, might be part of a larger protein structure, perhaps a solvent channel to the catalytic center. Therefore, they could play a role in substrate accessibility to it.     

Protein dynamics in cytochrome P450 molecular recognition and substrate specificity using 2D IR vibrational echo spectroscopy

Cytochrome (cyt) P450s hydroxylate a variety of substrates that can differ widely in their chemical structure. The importance of these enzymes in drug metabolism and other biological processes has motivated the study of the factors that enable their activity on diverse classes of molecules. Protein dynamics have been implicated in cyt P450 substrate specificity. Here, 2D IR vibrational echo spectroscopy is employed to measure the dynamics of cyt P450(cam) from Pseudomonas putida on fast time scales using CO bound at the active site as a vibrational probe. The substrate-free enzyme and the enzyme bound to both its natural substrate, camphor, and a series of related substrates are investigated to explicate the role of dynamics in molecular recognition in cyt P450(cam) and to delineate how the motions may contribute to hydroxylation specificity. In substrate-free cyt P450(cam), three conformational states are populated, and the structural fluctuations within a conformational state are relatively slow. Substrate binding selectively stabilizes one conformational state, and the dynamics become faster. Correlations in the observed dynamics with the specificity of hydroxylation of the substrates, the binding affinity, and the substrates' molecular volume suggest that motions on the hundreds of picosecond time scale contribute to the variation in activity of cyt P450(cam) toward different substrates.     

Chimeric streptogramin-tyrocidine antibiotics that overcome streptogramin resistance

Streptogramin antibiotics are comprised of two distinct chemical components: the type A polyketides and the type B cyclic depsipeptides. Clinical resistance to the type B streptogramins can occur via enzymatic degradation catalyzed by the lyase Vgb or by target modification through the action of Erm ribosomal RNA methyltransferases. We have prepared through chemical and chemo-enzymatic approaches a series of chimeric antibiotics composed of elements of type B streptogramins and the membrane-active antibiotic tyrocidine that evade these resistance mechanisms. These new compounds show broad antibiotic activity against gram-positive bacteria including a number of important pathogens, and chimeras appear to function by a mechanism that is distinct from their parent antibiotics. These results allow for the development of a brand new class of antibiotics with the ability to evade type B streptogramin-resistance mechanisms.     

Functional analysis of MycCI and MycG, cytochrome P450 enzymes involved in biosynthesis of mycinamicin macrolide antibiotics

Macrolides are a class of valuable antibiotics that include a macrolactone ring, at least one appended sugar unit, and, in most cases, additional hydroxyl or epoxide groups installed by cytochrome P450 enzymes. These functional groups contribute to structural diversification and serve to improve the bioactivity profiles of natural products. Here, we have characterized in vitro two P450 enzymes from the mycinamicin biosynthetic pathway of Micromonospora griseorubida. First, MycCI was characterized as the C21 methyl hydroxylase of mycinamicin VIII, the earliest macrolide form in the postpolyketide synthase tailoring pathway. Moreover, we established that optimal activity of MycCI depends on the native ferredoxin MycCII. Second, MycG P450 catalyzes consecutive hydroxylation and epoxidation reactions with mycinamicin IV as initial substrate. These reactions require prior dimethylation of 6-deoxyallose to mycinose for effective conversion by the dual function MycG enzyme.     

Insights into the importance of WPD-loop sequence for activity and structure in protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) possess a conserved mobile catalytic loop, the WPD-loop, which brings an aspartic acid into the active site where it acts as an acid/base catalyst. Prior experimental and computational studies, focused on the human enzyme PTP1B and the PTP from Yersinia pestis, YopH, suggested that loop conformational dynamics are important in regulating both catalysis and evolvability. We have generated a chimeric protein in which the WPD-loop of YopH is transposed into PTP1B, and eight chimeras that systematically restored the loop sequence back to native PTP1B. Of these, four chimeras were soluble and were subjected to detailed biochemical and structural characterization, and a computational analysis of their WPD-loop dynamics. The chimeras maintain backbone structural integrity, with somewhat slower rates than either wild-type parent, and show differences in the pH dependency of catalysis, and changes in the effect of Mg²⁺. The chimeric proteins'' WPD-loops differ significantly in their relative stability and rigidity. The time required for interconversion, coupled with electrostatic effects revealed by simulations, likely accounts for the activity differences between chimeras, and relative to the native enzymes. Our results further the understanding of connections between enzyme activity and the dynamics of catalytically important groups, particularly the effects of non-catalytic residues on key conformational equilibria.

Protein tyrosine phosphatases have a key catalytic residue on a mobile loop (the WPD-loop), making the connections between this loop sequence and its dynamics, together with the dynamics of other mobile loops, particularly important.     

A Minimal Functional Complex of Cytochrome P450 and FBD of Cytochrome P450 Reductase in Nanodiscs

Structural interactions that enable electron transfer to cytochrome‐P450 (CYP450) from its redox partner CYP450‐reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane‐bound functional complex to reveal interactions between the full‐length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function. Akin to cytochrome‐b₅ (cyt‐b₅), Arg 125 on the C‐helix of CYP450s is found to be important for effective electron transfer, thus supporting the competitive behavior of redox partners for CYP450s. A general approach is presented to study protein–protein interactions combining the use of nanodiscs with NMR spectroscopy and SAXS. Linking structural details to the mechanism will help unravel the xenobiotic metabolism of diverse microsomal CYP450s in their native environment and facilitate the design of new drug entities.     

Engineering cytochrome P-450s

Cytochrome P-450 isozymes represent a critical component of nature’s spectrum of detoxification catalysts that could be exploited for bioremediation. The ethanol-inducible human cytochrome P-450 2E1 serves as a model eukaryotic P-450 that complements the bacterial P-450 cam in dehalogenation and detoxification of environmental pollutants. We explored the construction of novel chimeric P-450s using cytochrome P-450 camC and 2E1 genes. For construction of chimera 1 (478 amino acids, 55.14 kDa), 145 amino acids from the N-terminus of P-450 2E1 protein (493 amino acids, 56.84 kDa) were replaced with 130 amino acids from the N-terminus of P-450 camC protein (415 amino acids, 46.66 kDa). In chimera 2 (525 amino acids, 60.24 kDa) the strategy involves replacement of 28 amino acids in the C-terminus of chimera 1 with 75 amino acids from the C-terminus of P-450 camC gene. Homology models of both the chimeric proteins were developed using SWISS-MODEL based on the known crystal structure of cytochrome P-450 camC, BM-3, 1DT6A, and 2C17A. The models indicated that the proposed heme-binding site was intact, which is inevitable for catalytic activity of cytochrome P-450s. The expression of chimera 1 and 2 genes in Escherichia coli DH5α was evident from light-pink cell pellets, protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis, and diagnostic carbon monoxide-difference spectra. Our studies show that strategies can be developed to exploit the natural diversity of the P-450 superfamily to generate chimeric biocatalysts that would provide new templates amenable to directed evolution.     

Thermodynamics and kinetics of PNA-DNA quadruplex-forming chimeras

PNA-DNA chimeras present the interesting properties of PNA, such as the high binding affinity to complementary single-strand (DNA or RNA), and the resistance to nuclease and protease degradation. At the same time, the limitations of an oligomer containing all PNA residues, such as low water solubility, self-aggregation, and low cellular uptake, are effectively overcome. Further, PNA-DNA chimeras possess interesting biological properties as antisense agents. We have explored the ability of PNA-DNA chimeric strands to assemble in quadruplex structures. The rate constant for association of the quadruplexes and their thermodynamic properties have been determined by CD spectroscopy and differential scanning calorimetry (DSC). Thermal denaturation experiments indicated higher thermal and thermodynamic stabilities for chimeric quadruplexes in comparison with the corresponding unmodified DNA quadruplex. Singular value decomposition analysis (SVD) suggests the presence of kinetically stable intermediate species in the quadruplex formation process. The experimental results have been discussed on the basis of molecular dynamic simulations. The ability of PNA-DNA chimeras to form stable quadruplex structures expands their potential utility as therapeutic agents.     

Carrier Protein-Free Enzymatic Biaryl Coupling in Arylomycin A2 Assembly and Structure of the Cytochrome P450 AryC**

The arylomycin antibiotics are potent inhibitors of bacterial type I signal peptidase. These lipohexapeptides contain a biaryl structural motif reminiscent of glycopeptide antibiotics. We herein describe the functional and structural evaluation of AryC, the cytochrome P450 performing biaryl coupling in biosynthetic arylomycin assembly. Unlike its enzymatic counterparts in glycopeptide biosynthesis, AryC converts free substrates without the requirement of any protein interaction partner, likely enabled by a strongly hydrophobic cavity at the surface of AryC pointing to the substrate tunnel. This activity enables chemo-enzymatic assembly of arylomycin A2 that combines the advantages of liquid- and solid-phase peptide synthesis with late-stage enzymatic cross-coupling. The reactivity of AryC is unprecedented in cytochrome P450-mediated biaryl construction in non-ribosomal peptides, in which peptidyl carrier protein (PCP)-tethering so far was shown crucial both in vivo and in vitro.     

Drug Oxidation by Cytochrome P450BM3: Metabolite Synthesis and Discovering New P450 Reaction Types

There is intense interest in late‐stage catalytic C?H bond functionalization as an integral part of synthesis. Effective catalysts must have a broad substrate range and tolerate diverse functional groups. Drug molecules provide a good test of these attributes of a catalyst. A library of P450_BM3 mutants developed from four base mutants with high activity for hydrocarbon oxidation produced human metabolites of a panel of drugs that included neutral (chlorzoxazone, testosterone), cationic (amitriptyline, lidocaine) and anionic (diclofenac, naproxen) compounds. No single mutant was active for all the tested drugs but multiple variants in the library showed high activity with each compound. The high conversions enabled full product characterization that led to the discovery of the new P450 reaction type of oxidative decarboxylation of an α‐hydroxy carboxylic acid and the formation a protected imine from an amine, offering a novel route to α‐functionalization of amines. The substrate range and varied product profiles suggest that this library of enzymes is a good basis for developing late‐stage C?H activation catalysts.     

Importance of C-Terminal Region for Thermostability of GH11 Xylanase from Streptomyces lividans

The amino acid sequences of xylanase B (XlnB) and xylanase C (XlnC) from Streptomyces lividans show significant homology. However, the temperature optima and stabilities of the two enzymes are quite different. XlnB exhibits an optimum temperature of 40 °C and retains 50% of its maximum activity at 43 °C, whereas the corresponding values for XlnC are 60 and 70 °C. To analyze these properties further, as well as to study the effect of the exchange of homologous segments in the C-terminal region, four chimeras designated as BSC, BFC, CSB, and CFB were constructed by substituting segments from the C-terminal homologous region of XlnB gene with that of XlnC and in turn substituting XlnC gene with that of XlnB. The purified chimeric enzymes were characterized with respect to pH/temperature activity, stability, and kinetic parameters. Most of enzymatic properties of chimeras were admixtures of those of the two parents. The chimeric enzymes were optimally active at 45–55 °C and pH 7.0. Both K _m and k _cat values of chimeric enzymes for p-nitrophenyl-β-d-cellobioside were admixtures of both parental enzymes, except that the k _cat value of chimeric BFC (2.79 s⁻¹) was higher than that of parental XlnC (1.99 s⁻¹). Notably, thermal stability of chimeric BSC and BFC was increased by 25 and 13 °C separately, as compared to one of parental XlnB, whereas the thermal stability of chimeric CSB and CFB was decreased by 23 and 21 °C, respectively, as compared to another parental XlnC. These results suggest that homologous C-terminal region in S. lividans GH11 xylanase appears to play an important role in determining enzyme characteristics, and exchanging of different segments of gene in this region might significantly alter or improve the enzymatic properties such as thermal stability.     

Association of Cytochrome P450 Enzymes is a Determining Factor in their Catalytic Activity

Previously, our laboratory demonstrated that one cytochrome P450 isoenzyme can influence the catalytic properties of another P450 isoenzyme when combined in a reconstituted system. Moreover, our data and that of other investigators indicate that P450 interaction is required for catalytic activity even when one isoenzyme is present. The goal of the current study was to examine the possible mechanism of these interactions in more detail. Analyzing recently published X-ray data of microsomal P450 enzymes and protein docking studies, four types of dimer formations of P450 enzymes were examined in more detail. In case of two dimer types, the aggregating partner was shown to contribute to NADPH cytochrome P450 reductase (CPR) binding-a flavoprotein whose interaction with P450 is required for expressing P450 functional activity of the neighboring P450 moiety. Thus, it was shown that dimerization of P450 enzymes might result in an altered affinity towards the CPR. Two dimer types were shown to exist only in the presence of a substrate, while the other two types exist also without a substrate present. The molecular basis was established for the fact that the presence of a substrate and other P450 enzymes simultaneously determine the catalytic activity. Furthermore, a kinetic model was improved describing the catalytic activity of P450 enzymes as a function of CPR concentration based on equilibrium between different supramolecular organizations of P450 enzymes. This model was successfully applied in order to explain our experimental data and that of other investigators.Eszter Hazai and Zsolt Bikádi contributed equally to this workDavid Kupfer-Deceased     

A High‐Throughput Mass Spectrometric Enzyme Activity Assay Enabling the Discovery of Cytochrome P450 Biocatalysts

Assaying for enzymatic activity is a persistent bottleneck in biocatalyst and drug development. Existing high‐throughput assays for enzyme activity tend to be applicable only to a narrow range of biochemical transformations, whereas universal enzyme characterization methods usually require chromatography to determine substrate turnover, greatly diminishing throughput. We present an enzyme activity assay that allows the high‐throughput mass‐spectrometric detection of enzyme activity in complex matrices without the need for a chromatographic step. This technology, which we call probing enzymes with click‐assisted NIMS (PECAN), can detect the activity of medically and biocatalytically significant cytochrome P450s in cell lysate, microsomes, and bacteria. Using this approach, a cytochrome P450_BM3 mutant library was successfully screened for the ability to catalyze the oxidation of the sesquiterpene valencene.     

The functional domains of dopamine transporter for cocaine analog, CFT binding

Cocaine analogue, CFT (2beta-carbomethoxy-3beta-(4-fluorophenyl) tropane) binding to dopamine transporter (DAT) in different species is quite heterogeneous. CFT is scarcely detected in bovine DAT whereas it is conspicuous in humans. To examine the structural basis for this functional discrepancy, we analyzed transporter chimeras of these two DATs. The CFT binding activities are avid in all of the chimeric DATs of which both of the 3rd and the 6-8th transmembrane domain (TM) are composed of human DAT sequences. On the contrary, CFT binding activities were scarcely detected if either or both of two regions are replaced with bovine sequences. These findings indicate that the CFT binding absolutely requires human DAT sequences, at least, in the regions encompassing the 3rd and 6-8th transmembrane domain (TM), and that these regions might contribute to form the 3-dimensional pocket for CFT binding.     

Substrate Specificity of Chimeric Enzymes Formed by Interchange of the Catalytic and Specificity Domains of the 5′-Nucleotidase UshA and the 3′-Nucleotidase CpdB

The 5′-nucleotidase UshA and the 3′-nucleotidase CpdB from Escherichia coli are broad-specificity phosphohydrolases with similar two-domain structures. Their N-terminal domains (UshA_Ndom and CpdB_Ndom) contain the catalytic site, and their C-terminal domains (UshA_Cdom and CpdB_Cdom) contain a substrate-binding site responsible for specificity. Both enzymes show only partial overlap in their substrate specificities. So, it was decided to investigate the catalytic behavior of chimeras bearing the UshA catalytic domain and the CpdB specificity domain, or vice versa. UshA_Ndom–CpdB_Cdom and CpdB_Ndom–UshA_Cdom were constructed and tested on substrates specific to UshA (5′-AMP, CDP-choline, UDP-glucose) or to CpdB (3′-AMP), as well as on 2′,3′-cAMP and on the common phosphodiester substrate bis-4-NPP (bis-4-nitrophenylphosphate). The chimeras did show neither 5′-nucleotidase nor 3′-nucleotidase activity. When compared to UshA, UshA_Ndom–CpdB_Cdom conserved high activity on bis-4-NPP, some on CDP-choline and UDP-glucose, and displayed activity on 2′,3′-cAMP. When compared to CpdB, CpdB_Ndom–UshA_Cdom conserved phosphodiesterase activities on 2′,3′-cAMP and bis-4-NPP, and gained activity on the phosphoanhydride CDP-choline. Therefore, the non-nucleotidase activities of UshA and CpdB are not fully dependent on the interplay between domains. The specificity domains may confer the chimeras some of the phosphodiester or phosphoanhydride selectivity displayed when associated with their native partners. Contrarily, the nucleotidase activity of UshA and CpdB depends strictly on the interplay between their native catalytic and specificity domains.     

Biphenyl Ether Analogs Containing Pomalidomide as Small-Molecule Inhibitors of the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Interaction

New biphenyl-based chimeric compounds containing pomalidomide were developed and evaluated for their activity to inhibit and degrade the programmed cell death-1/programmed cell death- ligand 1 (PD-1/PD-L1) complex. Most of the compounds displayed excellent inhibitory activity against PD-1/PD-L1, as assessed by the homogenous time-resolved fluorescence (HTRF) binding assay. Among them, compound 3 is one of the best with an IC₅₀ value of 60 nM. Using an ex vivo PD-1/PD-L1 blockade cell line bioassay that expresses human PD-1 and PD-L1, we show that compounds 4 and 5 significantly restore the repressed immunity in this co-culture model. Western blot data, however, demonstrated that these anti-PD-L1/pomalidomide chimeras could not reduce the protein levels of PD-L1.     

Bacterial Cytochrome P450 Catalyzed Post-translational Macrocyclization of Ribosomal Peptides**

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a fascinating group of natural products that exhibit diverse structural features and bioactivities. P450-catalyzed RiPPs stand out as a unique but underexplored family. Herein, we introduce a rule-based genome mining strategy that harnesses the intrinsic biosynthetic principles of RiPPs, including the co-occurrence and co-conservation of precursors and P450s and interactions between them, successfully facilitating the identification of diverse P450-catalyzed RiPPs. Intensive BGC characterization revealed four new P450s, KstB, ScnB, MciB, and SgrB, that can catalyze the formation of Trp-Trp-Tyr (one C−C and two C−N bonds), Tyr-Trp (C−C bond), Trp-Trp (C−N bond), and His-His (ether bond) crosslinks, respectively, within three or four residues. KstB, ScnB, and MciB could accept non-native precursors, suggesting they could be promising starting templates for bioengineering to construct macrocycles. Our study highlights the potential of P450s to expand the chemical diversity of strained macrocyclic peptides and the range of biocatalytic tools available for peptide macrocyclization.     

A phosphoramidate substrate analog is a competitive inhibitor of the Tetrahymena group I ribozyme

BACKGROUND: Phosphoramidate oligonucleotide analogs containing N3'-P5' linkages share many structural properties with natural nucleic acids and can be recognized by some RNA-binding proteins. Therefore, if the N-P bond is resistant to nucleolytic cleavage, these analogs may be effective substrate analog inhibitors of certain enzymes that hydrolyze RNA. We have explored the ability of the Tetrahymena group I intron ribozyme to bind and cleave DNA and RNA phosphoramidate analogs. RESULTS: The Tetrahymena group I ribozyme efficiently binds to phosphoramidate oligonucleotides but is unable to cleave the N3'-P5' bond. Although it adopts an A-form helical structure, the deoxyribo-phosphoramidate analog, like DNA, does not dock efficiently into the ribozyme catalytic core. In contrast, the ribo-phosphoramidate analog docks similarly to the native RNA substrate, and behaves as a competitive inhibitor of the group I intron 5' splicing reaction. CONCLUSIONS: Ribo-N3'-P5' phosphoramidate oligonucleotides are useful tools for structural and functional studies of ribozymes as well as protein-RNA interactions.