Mapping Potential Determinants of Peroxidative Activity in an Evolved Fungal Peroxygenase from Agrocybe aegerita

Fungal unspecific peroxygenases (UPOs) are hybrid biocatalysts with peroxygenative activity that insert oxygen into non-activated compounds, while also possessing convergent peroxidative activity for one electron oxidation reactions. In several ligninolytic peroxidases, the site of peroxidative activity is associated with an oxidizable aromatic residue at the protein surface that connects to the buried heme domain through a long-range electron transfer (LRET) pathway. However, the peroxidative activity of these enzymes may also be initiated at the heme access channel. In this study, we examined the origin of the peroxidative activity of UPOs using an evolved secretion variant (PaDa-I mutant) from Agrocybe aegerita as our point of departure. After analyzing potential radical-forming aromatic residues at the PaDa-I surface by QM/MM, independent saturation mutagenesis libraries of Trp24, Tyr47, Tyr79, Tyr151, Tyr265, Tyr281, Tyr293 and Tyr325 were constructed and screened with both peroxidative and peroxygenative substrates. These mutant libraries were mostly inactive, with only a few functional clones detected, none of these showing marked differences in the peroxygenative and peroxidative activities. By contrast, when the flexible Gly314-Gly318 loop that is found at the outer entrance to the heme channel was subjected to combinatorial saturation mutagenesis and computational analysis, mutants with improved kinetics and a shift in the pH activity profile for peroxidative substrates were found, while they retained their kinetic values for peroxygenative substrates. This striking change was accompanied by a 4.5°C enhancement in kinetic thermostability despite the variants carried up to four consecutive mutations. Taken together, our study proves that the origin of the peroxidative activity in UPOs, unlike other ligninolytic peroxidases described to date, is not dependent on a LRET route from oxidizable residues at the protein surface, but rather it seems to be exclusively located at the heme access channel.

Insights on intermolecular FMN-heme domain interaction and the role of linker length in cytochrome P450cin fusion proteins

Cytochrome P450s are an important group of enzymes catalyzing hydroxylation, and epoxidations reactions. In this work we describe the characterization of the CinA–CinC fusion enzyme system of a previously reported P450 using genetically fused heme (CinA) and FMN (CinC) enzyme domains from Citrobacter braaki. We observed that mixing individually inactivated heme (-) with FMN (-) domain in the CinA-10aa linker - CinC fusion constructs results in recovered activity and the formation of (2S)-2β-hydroxy,1,8-cineole (174 µM), a similar amount when compared to the fully functional fusion protein (176 µM). We also studied the effect of the fusion linker length in the activity complementation assay. Our results suggests an intermolecular interaction between heme and FMN parts from different CinA–CinC fusion protein similar to proposed mechanisms for P450 BM3 on the other hand, linker length plays a crucial influence on the activity of the fusion constructs. However, complementation assays show that inactive constructs with shorter linker lengths have functional subunits, and that the lack of activity might be due to incorrect interaction between fused enzymes.

Select amino acids in DGCR8 are essential for the UGU-pri-miRNA interaction and processing

Microprocessor, composed of DROSHA and DGCR8, processes primary microRNAs (pri-miRNAs) in miRNA biogenesis. Its cleavage efficiency and accuracy are enhanced because DGCR8 interacts with the apical UGU motif of pri-miRNAs. However, the mechanism and influence of DGCR8–UGU interaction on cellular miRNA expression are still elusive. In this study, we demonstrated that Rhed (i.e., the RNA-binding heme domain, amino acids 285–478) of DGCR8 interacts with UGU. In addition, we identified three amino acids 461–463 in Rhed, which are critical for the UGU interaction and essential for Microprocessor to accurately and efficiently process UGU-pri-miRNAs in vitro and UGU-miRNA expression in human cells. Furthermore, we found that within the DGCR8 dimer, the amino acids 461–463 from one monomer are capable of discriminating between UGU- and noUGU-pri-miRNAs. Our findings improve the current understanding of the substrate-recognizing mechanism of DGCR8 and implicate the roles of this recognition in differentiating miRNA expression in human cells.

Structures of microRNA-precursor apical junctions and loops reveal non-canonical base pairs important for processing

Metazoan pri-miRNAs and pre-miRNAs fold into characteristic hairpins that are recognized by the processing machinery. Essential to the recognition of these miR-precursors are their apical junctions where double-stranded stems meet single-stranded hairpin loops. Little is known about how apical junctions and loops fold in three-dimensional space. Here we developed a scaffold-directed crystallography method and determined the structures of eight human miR-precursor apical junctions and loops. Six structures contain non-canonical base pairs stacking on top of the hairpin stem. U-U pair contributes to thermodynamic stability in solution and is highly enriched at human miR-precursor apical junctions. Our systematic mutagenesis shows that U-U is among the most efficiently processed variants. The RNA-binding heme domain of pri-miRNA-processing protein DGCR8 binds longer loops more tightly and non-canonical pairs at the junction appear to modulate loop length. Our study provides structural and biochemical bases for understanding miR-precursors and molecular mechanisms of microRNA maturation.

Cryo-EM reveals the architecture of the dimeric cytochrome P450 CYP102A1 enzyme and conformational changes required for redox partner recognition

Cytochrome P450 family 102 subfamily A member 1 (CYP102A1) is a self-sufficient flavohemeprotein and a highly active bacterial enzyme capable of fatty acid hydroxylation at a >3,000 min−1 turnover rate. The CYP102A1 architecture has been postulated to be responsible for its extraordinary catalytic prowess. However, the structure of a functional full-length CYP102A1 enzyme remains to be determined. Herein, we used a cryo-EM single-particle approach, revealing that full-length CYP102A1 forms a homodimer in which both the heme and FAD domains contact each other. The FMN domain of one monomer was located close to the heme domain of the other monomer, exhibiting a trans configuration. Moreover, full-length CYP102A1 is highly dynamic, existing in multiple conformational states, including open and closed states. In the closed state, the FMN domain closely contacts the FAD domain, whereas in the open state, one of the FMN domains rotates away from its FAD domain and traverses to the heme domain of the other monomer. This structural arrangement and conformational dynamics may facilitate rapid intraflavin and trans FMN-to-heme electron transfers (ETs). Results with a variant having a 12-amino-acid deletion in the CYP102A1 linker region, connecting the catalytic heme and the diflavin reductase domains, further highlighted the importance of conformational dynamics in the ET process. Cryo-EM revealed that the Δ12 variant homodimer is conformationally more stable and incapable of FMN-to-heme ET. We conclude that closed-to-open alternation is crucial for redox partner recognition and formation of an active ET complex for CYP102A1 catalysis.