Mitochondrial contact site and cristae organizing system (MICOS) machinery supports heme biosynthesis by enabling optimal performance of ferrochelatase.

Heme is an essential cofactor required for a plethora of cellular processes in eukaryotes. In metazoans the heme biosynthetic pathway is typically partitioned between the cytosol and mitochondria, with the first and final steps taking place in the mitochondrion. The pathway has been extensively studied and its biosynthetic enzymes structurally characterized to varying extents. Nevertheless, understanding of the regulation of heme synthesis and factors that influence this process in metazoans remains incomplete. Therefore, we investigated the molecular organization as well as the physical and genetic interactions of the terminal pathway enzyme, ferrochelatase (Hem15), in the yeast Saccharomyces cerevisiae. Biochemical and genetic analyses revealed dynamic association of Hem15 with Mic60, a core component of the mitochondrial contact site and cristae organizing system (MICOS). Loss of MICOS negatively impacts Hem15 activity, affects the size of the Hem15 high-mass complex, and results in accumulation of reactive and potentially toxic tetrapyrrole precursors that may cause oxidative damage. Restoring intermembrane connectivity in MICOS-deficient cells mitigates these cytotoxic effects. These data provide new insights into how heme biosynthetic machinery is organized and regulated, linking mitochondrial architecture-organizing factors to heme homeostasis.

MinION sequencing to genotype US strains of infectious laryngotracheitis virus.

Over the last decade the US broiler industry has fought long-lasting outbreaks of infectious laryngotracheitis (ILTV). Previously, nine genotypes (I-IX) of ILTVs have been recognized using the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method with three viral alleles (gB, gM and UL47/gG). In this study, the genotyping system was simplified to six genotypes by amplicon sequencing and examining discriminating single nucleotide polymorphisms (SNPs) within these open reading frames. Using phylogenomic analysis of 27 full genomes of ILTV, a single allele (ORF A/ORF B) was identified containing SNPs that could differentiate ILTVs into genotypes congruent with the phylogenetic partitioning. The allelic variations allowed for the cataloging of the 27 strains into 5 genotypes: vaccinal TCO, vaccinal CEO, virulent CEO-like, virulent US and virulent US backyard flocks from 1980 to 1990, correlating with the PCR-RFLP genotypes I/ II/ III (TCO), IV (CEO), V (virulent CEO-like), VI (virulent US) and VII/VIII/IX (virulent US backyard flock isolates). With the unique capabilities of third generation sequencing, we investigated the application of Oxford Nanopore MinION technology for rapid sequencing of the amplicons generated in the single-allele assay. This technology was an improvement over Sanger-based sequencing of the single allele amplicons due to a booster amplification step in the MinION sequencing protocol. Overall, there was a 90% correlation between the genotyping results of the single-allele assay and the multi-allele assay. Surveillance of emerging ILTV strains could greatly benefit from real-time amplicon sequencing using the single-allele assay and MinION sequencing. RESEARCH HIGHLIGHTS A multi-allelic assay identified nine ILTV genotypes circulating in the US Single-allele genotyping is congruent with whole genome phylogenetic partitioning US ILTV strains can be grouped into five genotypes using the single-allele assay The single-allele assay can be done using MinION sequencing of barcoded amplicons.

Molecular characterization of the complete genome of falconid herpesvirus strain S-18.

Falconid herpesvirus type 1 (FaHV-1) is the causative agent of falcon inclusion body disease, an acute, highly contagious disease of raptors. The complete nucleotide sequence of the genome of FaHV-1 has been determined using Illumina MiSeq sequencing. The genome is 204,054 nucleotides in length and has a class E organization. The genome encodes approximately 130 putative protein-coding genes, of which 70 are orthologs of conserved alphaherpesvirus and Mardivirus proteins. Three FaHV-1 genes (UL3.5, UL44.5 and CIRC) were identified that encode protein homologues unique to Mardivirus and Varicellovirus. The genome also encodes homologues to the Mardivirus genes LORF2, LORF3, LORF4, LORF5, SORF3 and SORF4. An opal mutation resulting in premature termination was identified in the FaHV-1 UL43 gene. Phylogenetically, FaHV-1 resides in a monophyletic group with the other Mardiviruses but, along with anatid herpesvirus 1, represents a more distant divergence from the rest of the Mardivirus genus.

Identification and characterization of solvent-filled channels in human ferrochelatase.

Ferrochelatase catalyzes the formation of protoheme from two potentially cytotoxic products, iron and protoporphyrin IX. While much is known from structural and kinetic studies on human ferrochelatase of the dynamic nature of the enzyme during catalysis and the binding of protoporphyrin IX and heme, little is known about how metal is delivered to the active site and how chelation occurs. Analysis of all ferrochelatase structures available to date reveals the existence of several solvent-filled channels that originate at the protein surface and continue to the active site. These channels have been proposed to provide a route for substrate entry, water entry, and proton exit during the catalytic cycle. To begin to understand the functions of these channels, we investigated in vitro and in vivo a number of variants that line these solvent-filled channels. Data presented herein support the role of one of these channels, which originates at the surface residue H240, in the delivery of iron to the active site. Structural studies of the arginyl variant of the conserved residue F337, which resides at the back of the active site pocket, suggest that it not only regulates the opening and closing of active site channels but also plays a role in regulating the enzyme mechanism. These data provide insight into the movement of the substrate and water into and out of the active site and how this movement is coordinated with the reaction mechanism.

A pi-helix switch selective for porphyrin deprotonation and product release in human ferrochelatase.

Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) is the terminal enzyme in heme biosynthesis and catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX (heme). Due to the many critical roles of heme, synthesis of heme is required by the vast majority of organisms. Despite significant investigation of both the microbial and eukaryotic enzyme, details of metal chelation remain unidentified. Here we present the first structure of the wild-type human enzyme, a lead-inhibited intermediate of the wild-type enzyme with bound metallated porphyrin macrocycle, the product bound form of the enzyme, and a higher resolution model for the substrate-bound form of the E343K variant. These data paint a picture of an enzyme that undergoes significant changes in secondary structure during the catalytic cycle. The role that these structural alterations play in overall catalysis and potential protein-protein interactions with other proteins, as well as the possible molecular basis for these changes, is discussed. The atomic details and structural rearrangements presented herein significantly advance our understanding of the substrate binding mode of ferrochelatase and reveal new conformational changes in a structurally conserved pi-helix that is predicted to have a central role in product release.