Characterisation and engineering of a thermophilic RNA ligase from Palaeococcus pacificus.

RNA ligases are important enzymes in molecular biology and are highly useful for the manipulation and analysis of nucleic acids, including adapter ligation in next-generation sequencing of microRNAs. Thermophilic RNA ligases belonging to the RNA ligase 3 family are gaining attention for their use in molecular biology, for example a thermophilic RNA ligase from Methanobacterium thermoautotrophicum is commercially available for the adenylation of nucleic acids. Here we extensively characterise a newly identified RNA ligase from the thermophilic archaeon Palaeococcus pacificus (PpaRnl). PpaRnl exhibited significant substrate adenylation activity but low ligation activity across a range of oligonucleotide substrates. Mutation of Lys92 in motif I to alanine, resulted in an enzyme that lacked adenylation activity, but demonstrated improved ligation activity with pre-adenylated substrates (ATP-independent ligation). Subsequent structural characterisation revealed that in this mutant enzyme Lys238 was found in two alternate positions for coordination of the phosphate tail of ATP. In contrast mutation of Lys238 in motif V to glycine via structure-guided engineering enhanced ATP-dependent ligation activity via an arginine residue compensating for the absence of Lys238. Ligation activity for both mutations was higher than the wild-type, with activity observed across a range of oligonucleotide substrates with varying sequence and secondary structure.

The Pseudomonas syringae pv. actinidiae chemoreceptor protein F (PscF) periplasmic sensor domain: cloning, purification and X-ray crystallographic analysis.

Nitrate- and nitrite-sensing (NIT) domains are found associated with a wide variety of bacterial receptors, including chemoreceptors. However, the structure of a chemoreceptor-associated NIT domain has not yet been characterized. Recently, a chemoreceptor named PscF was identified from the plant pathogen Pseudomonas syringae pv. actinidiae that is predicted to contain a periplasmic NIT domain. The PscF sensor domain (PscF-SD; residues 42-332) was cloned into an appropriate expression vector, recombinantly produced in Escherichia coli BL21-Gold(DE3) cells and purified via immobilized metal-affinity and size-exclusion chromatography. Purified PscF-SD was screened for crystallization; the best crystal diffracted to a maximum resolution of 1.46 Šin space group P212121. However, the data could not be phased using the only available NIT-domain structure (Klebsiella oxytoca NasR; PDB entry 4akk) as the search model. Therefore, a data set from a selenomethionine-labelled protein crystal was also collected. The selenomethionine-labelled protein crystal diffracted to a resolution of 2.46 Šin space group P212121. These data will be used to attempt to solve the structure using the single-wavelength anomalous diffraction technique. The structure is expected to provide insights into the ligand specificity of NIT domains and the role of NIT domains in chemotaxis.

Structures and kinetics for plant nucleoside triphosphate diphosphohydrolases support a domain motion catalytic mechanism.

Extracellular nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes that hydrolyze extracellular nucleotides to the respective monophosphate nucleotides. In the past 20 years, NTPDases belonging to mammalian, parasitic and prokaryotic domains of life have been discovered, cloned and characterized. We reveal the first structures of NTPDases from the legume plant species Trifolium repens (7WC) and Vigna unguiculata subsp. cylindrica (DbLNP). Four crystal structures of 7WC and DbLNP were determined at resolutions between 1.9 and 2.6 Å. For 7WC, structures were determined for an -apo form (1.89 Å) and with the product AMP (2.15 Å) and adenine and phosphate (1.76 Å) bound. For DbLNP, a structure was solved with phosphate and manganese bound (2.60 Å). Thorough kinetic data and analysis is presented. The structure of 7WC and DbLNP reveals that these NTPDases can adopt two conformations depending on the molecule and co-factor bound in the active site. A central hinge region creates a "butterfly-like" motion of the domains that reduces the width of the inter-domain active site cleft upon molecule binding. This phenomenon has been previously described in Rattus norvegicus and Legionella pneumophila NTPDaseI and Toxoplasma gondii NTPDaseIII suggesting a common catalytic mechanism across the domains of life.