We report the first characterization and classification of Orf13 (S. refuineus) as a heme dependent peroxidase catalyzing the ortho-hydroxylation of l-tyrosine to l-DOPA. The putative tyrosine hydroxylase coded by orf13 of the anthramycin biosynthesis gene cluster has been expressed and purified. Heme b has been identified as the required cofactor for catalysis and maximal l-tyrosine conversion to l-DOPA is observed in the presence of hydrogen peroxide. Pre-incubation of l-tyrosine with Orf13 prior to the addition of hydrogen peroxide is required for l-DOPA production. However, the enzyme becomes inactivated by hydrogen peroxide during catalysis. Steady state kinetic analysis of l-tyrosine hydroxylation revealed similar catalytic efficiency for both l-tyrosine and hydrogen peroxide. Spectroscopic data from a reduced-CO (g) UV-visible spectrum of Orf13 and electron paramagnetic resonance of ferric-heme Orf13 are consistent with heme peroxidases that have a histidyl-ligated heme-iron. Contrary to the classical heme peroxidase oxidation reaction with hydrogen peroxide that produces coupled aromatic products such as o,o'-dityrosine, Orf13 is novel in its ability to catalyze aromatic amino acid hydroxylation with hydrogen peroxide, in the substrate addition order and for its substrate specificity for l-tyrosine. Peroxygenase activity of Orf13 for the ortho-hydroxylation of l-tyrosine to l-DOPA by a molecular oxygen dependent pathway in the presence of dihydroxyfumaric acid is also observed. This reaction behavior is consistent with peroxygenase activity reported with horseradish peroxidase for the hydroxylation of phenol. Overall, the putative function of Orf13 as a tyrosine hydroxylase has been confirmed and establishes the first bacterial class of tyrosine hydroxylases.

We present high resolution single cell profiling of tumor infiltrating lymphocytes (TILs) for immune research and cancer target discovery. TILs hold the key to understanding anti-cancer immune responses but remain challenging to study due to their vast range of phenotypes, functions and abundance. Current approaches to TIL analysis such as immune receptor sequencing, flow cytometry, and RNA expression profiling are limited, since each approach gives only partial insights into a TIL repertoire and their results cannot be connected at the single cell level. We therefore built a technology for simultaneous determination of immune receptor sequences, surface protein phenotypes and RNA expression profiles from single TILs at unprecedented scale. Able to process millions of cells per experiment, the throughput of our emulsion-based DNA barcoding method allows deep and unbiased TIL profiling directly from tumor tissue without the need for cell sorting, stimulation or culture. Briefly, single-cell dissociated biopsy tissue samples are stained with DNA-labeled surface marker antibodies and isolated into individual ∼65-pL microdroplets by a microfluidic emulsion chip. Within the droplets, cellular RNAs are reverse transcribed into cDNA, and a barcoding step attaches droplet-specific DNA barcodes to all of the cDNAs as well as to the DNA labels carried by the marker antibodies. At the end of the reaction, all products associated with a single input cell carry the same DNA barcode sequence, allowing subsequent high throughput sequencing to assign the products to their cell of origin. During analysis the full-length immune receptor pairs for each B- and T-cell are reconstructed and the marker antibody tags are identified and quantified along with dozens of additional mRNA markers, resulting in a high-dimensional dataset containing integrated immune receptor clone, protein marker and RNA expression profiles of thousands to hundreds of thousands of individual lymphocytes in the input sample. We have successfully applied our method to a variety of healthy blood and tissue samples including human cancers such as pancreatic, ovarian and lung cancer, profiling millions of B-cells and T-cells present in the samples. We identify likely tumor-reactive TILs by finding BCR or TCR clonal lineages associated with particular protein and RNA signatures, including expression of immunosuppression markers such as PD-1 and CTLA-4. Following candidate selection, full-length BCR and TCR pairs are synthesized and expressed for functional screening and target antigen identification. By providing an unprecedented view into TIL diversity across a wide range of disease and sample types, our platform also has widespread potential for basic and applied research into the patient immune response to cancer and the dynamics of immunotherapy. Citation Format: Katherine L. Connor, Adrian W. Briggs, Stephen J. Goldfless, Sonia Timberlake, Brian J. Belmont, Christopher R. Clouser, David Koppstein, Francois Vigneault. Comprehensive TIL profiling by simultaneous DNA barcoding of proteins, RNA and natively paired immune receptors from millions of single cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1442.