Virus-encoded glycosyltransferases hypermodify DNA with diverse glycans.

Enzymatic modification of DNA nucleobases can coordinate gene expression, nuclease protection, or mutagenesis. We recently discovered a clade of phage-specific cytosine methyltransferase (MT) and 5-methylpyrimidine dioxygenase (5mYOX) enzymes that produce 5-hydroxymethylcytosine (5hmC) as a precursor for enzymatic hypermodifications on viral genomes. Here, we identify phage MT- and 5mYOX-associated glycosyltransferases (GTs) that catalyze linkage of diverse sugars to 5hmC nucleobase substrates.

A roadmap for the functional annotation of protein families: a community perspective.

Over the last 25 years, biology has entered the genomic era and is becoming a science of 'big data'. Most interpretations of genomic analyses rely on accurate functional annotations of the proteins encoded by more than 500 000 genomes sequenced to date. By different estimates, only half the predicted sequenced proteins carry an accurate functional annotation, and this percentage varies drastically between different organismal lineages.

Phage-encoded ten-eleven translocation dioxygenase (TET) is active in C5-cytosine hypermodification in DNA.

TET/JBP (ten-eleven translocation/base J binding protein) enzymes are iron(II)- and 2-oxo-glutarate-dependent dioxygenases that are found in all kingdoms of life and oxidize 5-methylpyrimidines on the polynucleotide level. Despite their prevalence, few examples have been biochemically characterized. Among those studied are the metazoan TET enzymes that oxidize 5-methylcytosine in DNA to hydroxy, formyl, and carboxy forms and the euglenozoa JBP dioxygenases that oxidize thymine in the first step of base J biosynthesis.

The surprising structural and mechanistic dichotomy of membrane-associated phosphoglycosyl transferases.

Phosphoglycosyl transferases (PGTs) play a pivotal role at the inception of complex glycoconjugate biosynthesis pathways across all domains of life. PGTs promote the first membrane-committed step in the en bloc biosynthetic strategy by catalyzing the transfer of a phospho-sugar from a nucleoside diphospho-sugar to a membrane-resident polyprenol phosphate. Studies on the PGTs have been hampered because they are integral membrane proteins, and often prove to be recalcitrant to expression, purification and analysis.

Glycoconjugate pathway connections revealed by sequence similarity network analysis of the monotopic phosphoglycosyl transferases.

The monotopic phosphoglycosyl transferase (monoPGT) superfamily comprises over 38,000 nonredundant sequences represented in bacterial and archaeal domains of life. Members of the superfamily catalyze the first membrane-committed step in en bloc oligosaccharide biosynthetic pathways, transferring a phosphosugar from a soluble nucleoside diphosphosugar to a membrane-resident polyprenol phosphate. The singularity of the monoPGT fold and its employment in the pivotal first membrane-committed step allows confident assignment of both protein and corresponding pathway.

Structural Analysis of Binding Determinants of Trehalose-6-phosphate Phosphatase Using Ground-State Complexes.

Trehalose-6-phosphate phosphatase (T6PP) catalyzes the dephosphorylation of trehalose 6-phosphate (T6P) to the disaccharide trehalose. The enzyme is not present in mammals but is essential to the viability of multiple lower organisms as trehalose is a critical metabolite, and T6P accumulation is toxic. Hence, T6PP is a target for therapeutics of human pathologies caused by bacteria, fungi, and parasitic nematodes.

Lanthanide-Binding Tags for 3D X-ray Imaging of Proteins in Cells at Nanoscale Resolution.

We report the application of lanthanide-binding tags (LBTs) for two- and three-dimensional X-ray imaging of individual proteins in cells with a sub-15 nm beam. The method combines encoded LBTs, which are tags of minimal size (ca. 15-20 amino acids) affording high-affinity lanthanide ion binding, and X-ray fluorescence microscopy (XFM).

X-ray Fluorescence Nanotomography of Single Bacteria with a Sub-15 nm Beam.

X-ray Fluorescence (XRF) microscopy is a growing approach for imaging the trace element concentration, distribution, and speciation in biological cells at the nanoscale. Moreover, three-dimensional nanotomography provides the added advantage of imaging subcellular structure and chemical identity in three dimensions without the need for staining or sectioning of cells. To date, technical challenges in X-ray optics, sample preparation, and detection sensitivity have limited the use of XRF nanotomography in this area.

Crystallization of Liganded Phosphatases in the HAD Superfamily.

Phosphotransferases catalyze reactions on chemically diverse molecules in organisms from all domains of life. The haloalkanoate dehalogenase superfamily (HADSF) is a model system for phosphoryl transfer enzymes as members catalyze phosphoester hydrolase, phosphonate hydrolase, and phosphomutase reactions on sugars, lipids, nucleotides, and peptides. Because these reactions are fundamental to essential metabolic transformations, understanding the mechanism and determinants of substrate specificity in the HADSF is critical.