Non-ribosomal peptide synthetase (NRPS) mega-enzyme complexes are modular assembly lines that are involved in the biosynthesis of numerous peptide metabolites independently of the ribosome. The multiple interactions between catalytic domains within the NRPS machinery are further complemented by additional interactions with external enzymes, particularly focused on the final peptide maturation process. An important class of NRPS metabolites that require extensive external modification of the NRPS-bound peptide are the glycopeptide antibiotics (GPAs), which include vancomycin and teicoplanin. These clinically relevant peptide antibiotics undergo cytochrome P450-catalysed oxidative crosslinking of aromatic side chains to achieve their final, active conformation. However, the mechanism underlying the recruitment of the cytochrome P450 oxygenases to the NRPS-bound peptide was previously unknown. Here we show, through in vitro studies, that the X-domain, a conserved domain of unknown function present in the final module of all GPA NRPS machineries, is responsible for the recruitment of oxygenases to the NRPS-bound peptide to perform the essential side-chain crosslinking. X-ray crystallography shows that the X-domain is structurally related to condensation domains, but that its amino acid substitutions render it catalytically inactive. We found that the X-domain recruits cytochrome P450 oxygenases to the NRPS and determined the interface by solving the structure of a P450-X-domain complex. Additionally, we demonstrated that the modification of peptide precursors by oxygenases in vitro--in particular the installation of the second crosslink in GPA biosynthesis--occurs only in the presence of the X-domain. Our results indicate that the presentation of peptidyl carrier protein (PCP)-bound substrates for oxidation in GPA biosynthesis requires the presence of the NRPS X-domain to ensure conversion of the precursor peptide into a mature aglycone, and that the carrier protein domain alone is not always sufficient to generate a competent substrate for external cytochrome P450 oxygenases.

Bacterial cytochrome P450s form a remarkable clade of the P450 superfamily of oxidative hemoproteins, and are often involved in the biosynthesis of complex natural products. Those in a subgroup known as "Oxy enzymes" play a crucial role in the biosynthesis of glycopeptide antibiotics, including vancomycin and teicoplanin. The Oxy enzymes catalyze crosslinking of aromatic residues in the non-ribosomal antibiotic precursor peptide while it remains bound to the non-ribosomal peptide synthetase (NRPS); this crosslinking secures the three-dimensional structure of the glycopeptide, crucial for antibiotic activity. We have characterized OxyBtei , the first of the Oxy enzymes in teicoplanin biosynthesis. Our results reveal that OxyBtei possesses a structure similar to those of other Oxy proteins and is active in crosslinking NRPS-bound peptide substrates. However, OxyBtei displays a significantly altered activity spectrum against peptide substrates compared to its well-studied vancomycin homologue.

Electrostatic interactions between the positively and negatively charged amino acids in proteins play an important role in macromolecular stability, binding, and recognition. Numerous amino acids in proteins are ionizable and may exist in negatively (e.g., Glu, Asp, Cys, Tyr) or positively (e.g., Arg, Lys, His, Orn) charged form dependent on pH and their pKas. In this work, isothermal titration calorimetry was used to determine the average standard values of thermodynamic parameters (the Gibbs free energy, enthalpy, entropy, and the heat capacity) of interaction between the positively charged amino acid homopolymers (polyarginine, polylysine, and polyornithine) and the negatively charged homopolymers (polyaspartic and polyglutamic acids). These values are of potential use in the computational models of interacting proteins and other biological macromolecules. The study showed that oppositely charged poly(amino acid)s bound each other with the stoichiometry of one positive to one negative charge. Arginine bound to the negatively charged amino acids with exothermic enthalpy and higher affinity than lysine. This result also suggests that positive charges in proteins should not be considered entirely equivalent if carried by lysine or arginine. The difference in binding energy of arginine and lysine association with the negatively charged amino acids was attributed to the enthalpy of the second ionic hydrogen bond formation between the guanidine and carboxylic groups. Despite the favorable enthalpic contribution, all such ion pair formation reactions were largely entropy-driven. Consistent with previously observed ionic interactions, the positive heat capacity was always observed during the amino acid ion pair formation.

Pyrimidine dimers are the most common DNA lesions generated under UV radiation. To reveal the molecular mechanisms behind their formation, it is of significance to reveal the roles of each pyrimidine residue. We thus replaced the 5'-pyrimidine residue with a photochemically inert xylene moiety (X). The electron-rich X can be readily oxidized but not reduced defining the direction of interbase electron transfer (ET). Irradiation of the XpT dinucleotide under 254 nm UV light generates two major photoproducts: a pyrimidine (6-4) pyrimidone (6-4PP) analog and an analog of the socalled spore photoproduct (SP). Both products are formed via reaction at C4=O of the photo-excited 3'-T, which indicates that excitation of a single "driver" residue is sufficient to trigger pyrimidine dimerization. Our quantum-chemical calculations demonstrated that photo-excited 3'-T accepts an electron from 5′-X. The resulting charge-separated radical pair lowers its energy upon formation of interbase covalent bonds eventually yielding 6-4PP and SP.

Photoswitching of phytochrome photoreceptors between red-absorbing (Pr) and far-red absorbing (Pfr) states triggers light adaptation of plants, bacteria and other organisms. Using quantum chemistry, we elucidate the color-tuning mechanism of phytochromes and identify the origin of the Pfr-state red-shifted spectrum. Spectral variations are explained by resonance interactions of the protonated linear tetrapyrrole chromophore. In particular, hydrogen bonding of pyrrole ring D with the strictly conserved aspartate shifts the positive charge towards ring D thereby inducing the red spectral shift. Our MD simulations demonstrate that formation of the ring D–aspartate hydrogen bond depends on interactions between the chromophore binding domain (CBD) and phytochrome specific domain (PHY). Our study guides rational engineering of fluorescent phytochromes with a far-red shifted spectrum.