luxA Gene From Enhygromyxa salina Encodes a Functional Homodimeric Luciferase.

Several clades of luminescent bacteria are known currently. They all contain similar lux operons, which include the genes luxA and luxB encoding a heterodimeric luciferase. The aldehyde oxygenation reaction is presumed to be catalyzed primarily by the subunit LuxA, whereas LuxB is required for efficiency and stability of the complex. Recently, genomic analysis identified a subset of bacterial species with rearranged lux operons lacking luxB. Here, we show that the product of the luxA gene from the reduced luxACDE operon of Enhygromyxa salina is luminescent upon addition of aldehydes both in vivo in Escherichia coli and in vitro. Overall, EsLuxA is much less bright compared with luciferases from Aliivibrio fischeri (AfLuxAB) and Photorhabdus luminescens (PlLuxAB), and most active with medium-chain C4-C9 aldehydes. Crystal structure of EsLuxA determined at the resolution of 2.71 Å reveals a (ß/α)8 TIM-barrel fold, characteristic for other bacterial luciferases, and the protein preferentially forms a dimer in solution. The mobile loop residues 264-293, which form a ß-hairpin or a coil in Vibrio harveyi LuxA, form α-helices in EsLuxA. Phylogenetic analysis shows EsLuxA and related proteins may be bacterial protoluciferases that arose prior to duplication of the luxA gene and its speciation to luxA and luxB in the previously described luminescent bacteria. Our work paves the way for the development of new bacterial luciferases that have an advantage of being encoded by a single gene.

Structural insights into thrombolytic activity of destabilase from medicinal leech.

Destabilase from the medical leech Hirudo medicinalis belongs to the family of i-type lysozymes. It has two different enzymatic activities: microbial cell walls destruction (muramidase activity), and dissolution of the stabilized fibrin (isopeptidase activity). Both activities are known to be inhibited by sodium chloride at near physiological concentrations, but the structural basis remains unknown. Here we present two crystal structures of destabilase, including a 1.1 Å-resolution structure in complex with sodium ion. Our structures reveal the location of sodium ion between Glu34/Asp46 residues, which were previously recognized as a glycosidase active site. While sodium coordination with these amino acids may explain inhibition of the muramidase activity, its influence on previously suggested Ser49/Lys58 isopeptidase activity dyad is unclear. We revise the Ser49/Lys58 hypothesis and compare sequences of i-type lysozymes with confirmed destabilase activity. We suggest that the general base for the isopeptidase activity is His112 rather than Lys58. pKa calculations of these amino acids, assessed through the 1 µs molecular dynamics simulation, confirm the hypothesis. Our findings highlight the ambiguity of destabilase catalytic residues identification and build foundations for further research of structure-activity relationship of isopeptidase activity as well as structure-based protein design for potential anticoagulant drug development.

Nitrate- and Nitrite-Sensing Histidine Kinases: Function, Structure, and Natural Diversity.

Under anaerobic conditions, bacteria may utilize nitrates and nitrites as electron acceptors. Sensitivity to nitrous compounds is achieved via several mechanisms, some of which rely on sensor histidine kinases (HKs). The best studied nitrate- and nitrite-sensing HKs (NSHKs) are NarQ and NarX from Escherichia coli. Here, we review the function of NSHKs, analyze their natural diversity, and describe the available structural information. In particular, we show that around 6000 different NSHK sequences forming several distinct clusters may now be found in genomic databases, comprising mostly the genes from Beta- and Gammaproteobacteria as well as from Bacteroidetes and Chloroflexi, including those from anaerobic ammonia oxidation (annamox) communities. We show that the architecture of NSHKs is mostly conserved, although proteins from Bacteroidetes lack the HAMP and GAF-like domains yet sometimes have PAS. We reconcile the variation of NSHK sequences with atomistic models and pinpoint the structural elements important for signal transduction from the sensor domain to the catalytic module over the transmembrane and cytoplasmic regions spanning more than 200 Å.