Predominant Biphenyl Dioxygenase From Legacy Polychlorinated Biphenyl (PCB)-Contaminated Soil Is a Part of Unusual Gene Cluster and Transforms Flavone and Flavanone

In this study, the diversity of bphA genes was assessed in a 13C-enriched metagenome upon stable isotope probing (SIP) of microbial populations in legacy PCB-contaminated soil with 13C-biphenyl (BP). In total, 13 bphA sequence variants (SVs) were identified in the final amplicon dataset. Of these, one SV comprised 59% of all sequences, and when it was translated into a protein sequence, it exhibited 87, 77.4, and 76.7% identity to its homologs from Pseudomonas furukawaii KF707, Cupriavidus sp. WS, and Pseudomonas alcaliphila B-367, respectively. This same BphA sequence also contained unusual amino acid residues, Alanine, Valine, and Serine in region III, which had been reported to be crucial for the substrate specificity of the corresponding biphenyl dioxygenase (BPDO), and was accordingly designated BphA_AVS. The DNA locus of 18 kbp containing the BphA_AVS-coding sequence retrieved from the metagenome was comprised of 16 ORFs and was most likely borne by Paraburkholderia sp. The BPDO corresponding to bphAE_AVS was cloned and heterologously expressed in E. coli, and its substrate specificity toward PCBs and a spectrum of flavonoids was assessed. Although depleting a rather narrow spectrum of PCB congeners, the efficient transformation of flavone and flavanone was demonstrated through dihydroxylation of the B-ring of the molecules. The homology-based functional assignment of the putative proteins encoded by the rest of ORFs in the AVS region suggests their potential involvement in the transformation of aromatic compounds, such as flavonoids. In conclusion, this study contributes to the body of information on the involvement of soil-borne BPDOs in the metabolism of flavonoid compounds, and our paper provides a more advanced context for understanding the interactions between plants, microbes and anthropogenic compounds in the soil.

Scalable Synthesis of L-allo-Enduracididine: The Unusual Amino Acid Present in Teixobactin

A scalable synthesis of L-allo-enduracididine is achieved from commercially available (S)-glycidol in ten linear steps involving well established synthetic transformations. The synthetic route is flexible and can be used to synthesize all four diastereomers, by changing the stereochemistry of glycidol and Sharpless asymmetric dihydroxylation reagent.

Doubly Protonated Species Collision Induced Dissociation for Identification of Isocyclosporins

Nonribosomal cyclopeptide cyclosporin A (CsA), produced by fungus <i>Tolypocladium inflatum</i>, is an extremely important immunosuppressive drug used in organ transplantations and for therapy of autoimmune diseases. Here we report for the first time production of CsA, along with related cyclosporins B and C, by <i>Tolypocladium inflatum </i>strains of marine origin (White Sea). Cyclosporins A–C contain an unusual amino acid, (4<i>R</i>)-4-((<i>E</i>)-2-butenyl)-4,<i>N</i>-dimethyl-l-threonine (MeBmt), and are prone to isomerization to non-active isocyclosporine by N→O acyl shift of valine connected to MeBmt in acidic conditions. CsA and isoCsA are not distinguishable in MS analysis of [M+H]⁺ ions due to the rapid [CsA+H]⁺→[isoCsA+H]⁺ conversion. We found that the N→O acyl shift is completely suppressed in cyclosporine [M+2H]²⁺ ions, and their MS/MS fragmentation can be used for rapid and unambiguous analysis of cyclosporins and isocylosporins. The fragmentation patterns of [CyA+2H]²⁺ and [isoCyA+2H]²⁺ ions were analyzed and explained. The developed approach could be useful for MS analysis of other peptides containing β-hydroxy-α-amino acids.

Doubly Protonated Species Collision Induced Dissociation for Identification of Isocyclosporins

Nonribosomal cyclopeptide cyclosporin A (CsA), produced by fungus <i>Tolypocladium inflatum</i>, is an extremely important immunosuppressive drug used in organ transplantations and for therapy of autoimmune diseases. Here we report for the first time production of CsA, along with related cyclosporins B and C, by <i>Tolypocladium inflatum </i>strains of marine origin (White Sea). Cyclosporins A–C contain an unusual amino acid, (4<i>R</i>)-4-((<i>E</i>)-2-butenyl)-4,<i>N</i>-dimethyl-l-threonine (MeBmt), and are prone to isomerization to non-active isocyclosporine by N→O acyl shift of valine connected to MeBmt in acidic conditions. CsA and isoCsA are not distinguishable in MS analysis of [M+H]⁺ ions due to the rapid [CsA+H]⁺→[isoCsA+H]⁺ conversion. We found that the N→O acyl shift is completely suppressed in cyclosporine [M+2H]²⁺ ions, and their MS/MS fragmentation can be used for rapid and unambiguous analysis of cyclosporins and isocylosporins. The fragmentation patterns of [CyA+2H]²⁺ and [isoCyA+2H]²⁺ ions were analyzed and explained. The developed approach could be useful for MS analysis of other peptides containing β-hydroxy-α-amino acids.

An Unusual Amino Acid Substitution Within Hummingbird Cytochrome c Oxidase Alters a Key Proton-Conducting Channel

Hummingbirds in flight exhibit the highest mass-specific metabolic rate of all vertebrates. The bioenergetic requirements associated with sustained hovering flight raise the possibility of unique amino acid substitutions that would enhance aerobic metabolism. Here, we have identified a non-conservative substitution within the mitochondria-encoded cytochrome c oxidase subunit I (COI) that is fixed within hummingbirds, but not among other vertebrates. This unusual change is also rare among metazoans, but can be identified in several clades with diverse life histories. We performed atomistic molecular dynamics simulations using bovine and hummingbird COI models, thereby bypassing experimental limitations imposed by the inability to modify mtDNA in a site-specific manner. Intriguingly, our findings suggest that COI amino acid position 153 (bovine numbering convention) provides control over the hydration and activity of a key proton channel in COX. We discuss potential phenotypic outcomes linked to this alteration encoded by hummingbird mitochondrial genomes.

An unusual amino acid substitution within hummingbird cytochrome c oxidase alters a key proton-conducting channel

ABSTRACTHummingbirds in flight exhibit the highest metabolic rate of all vertebrates. The bioenergetic requirements associated with sustained hovering flight raise the possibility of unique amino acid substitutions that would enhance aerobic metabolism. Here, we have identified a non-conservative substitution within the mitochondria-encoded cytochrome c oxidase subunit I (COI) that is fixed within hummingbirds, but not among other vertebrates. This unusual change is also rare among metazoans, but can be identified in several clades with diverse life histories. We performed atomistic molecular dynamics simulations using bovine and hummingbird COI models, thereby bypassing experimental limitations imposed by the inability to modify mtDNA in a site-specific manner. Intriguingly, our findings suggest that COI amino acid position 153 (bovine numbering system) provides control over the hydration and activity of a key proton channel in COX. We discuss potential phenotypic outcomes linked to this alteration encoded by the hummingbird mitochondrial genome.

Crystal structures of pyrrolidone-carboxylate peptidase I from Deinococcus radiodurans reveal the mechanism of L-pyroglutamate recognition

Pyrrolidone-carboxylate peptidase (PCP) catalyzes the removal of an unusual amino acid, L-pyroglutamate (pG), from the N-termini of peptides and proteins. It has implications in the functional regulation of different peptides in both prokaryotes and eukaryotes. However, the pG-recognition mechanism of the PCP enzyme remains largely unknown. Here, crystal structures of PCP I from Deinococcus radiodurans (PCPdr) are reported in pG-free and pG-bound forms at resolutions of 1.73 and 1.55 Å, respectively. Four protomers in PCPdr form a tetrameric structure. The residues responsible for recognizing the pG residue are mostly contributed by a flexible loop (loop A) that is present near the active site. These residues are conserved in all known PCPs I, including those from mammals. Phe9 and Phe12 of loop A form stacking interactions with the pyrrolidone ring of pG, while Asn18 forms a hydrogen bond to OE of pG. The main chain of a nonconserved residue, Leu71, forms two hydrogen bonds to NH and OE of pG. Thus, pG is recognized in the S1 substrate subsite of the enzyme by both van der Waals and polar interactions, which provide specificity for the pG residue of the peptide. In contrast to previously reported PCP I structures, the PCPdr tetramer is in a closed conformation with an inaccessible active site. The structures show that the active site can be accessed by the substrates via disordering of loop A. This disordering could also prevent product inhibition by releasing the bound pG product from the S1 subsite, thus allowing the enzyme to engage a fresh substrate.