Mechanism of mycolic acid cyclopropane synthase: a theoretical study

The reaction mechanism of mycolic acid cyclopropane synthase is investigated using hybrid density functional theory. The direct methylation mechanism is examined with a large model of the active site constructed on the basis of the crystal structure of the native enzyme. The important active site residue Glu140 is modeled in both ionized and neutral forms. We demonstrate that the reaction starts via the transfer of a methyl to the substrate double bond, followed by the transfer of a proton from the methyl cation to the bicarbonate present in the active site. The first step is calculated to be rate-limiting, in agreement with experimental kinetic results. The protonation state of Glu140 has a rather weak influence on the reaction energetics. In addition to the natural reaction, a possible side reaction, namely a carbocation rearrangement, is also considered and is shown to have a low barrier. Finally, the energetics for the sulfur ylide proposal, which has already been ruled out, is also estimated, showing a large energetic penalty for ylide formation.     

The non-canonical protein binding site at the monomer-monomer interface of yeast proliferating cell nuclear antigen (PCNA) regulates the Rev1-PCNA interaction and Polζ/Rev1-dependent translesion DNA synthesis

Rev1 and DNA polymerase ζ (Polζ) are involved in the tolerance of DNA damage by translesion synthesis (TLS). The proliferating cell nuclear antigen (PCNA), the auxiliary factor of nuclear DNA polymerases, plays an important role in regulating the access of TLS polymerases to the primer terminus. Both Rev1 and Polζ lack the conserved hydrophobic motif that is used by many proteins for the interaction with PCNA at its interdomain connector loop. We have previously reported that the interaction of yeast Polζ with PCNA occurs at an unusual site near the monomer-monomer interface of the trimeric PCNA. Using GST pull-down assays, PCNA-coupled affinity beads pull-down and gel filtration chromatography, we show that the same region is required for the physical interaction of PCNA with the polymerase-associated domain (PAD) of Rev1. The interaction is disrupted by the pol30-113 mutation that results in a double amino acid substitution at the monomer-monomer interface of PCNA. Genetic analysis of the epistatic relationship of the pol30-113 mutation with an array of DNA repair and damage tolerance mutations indicated that PCNA-113 is specifically defective in the Rev1/Polζ-dependent TLS pathway. Taken together, the data suggest that Polζ and Rev1 are unique among PCNA-interacting proteins in using the novel binding site near the intermolecular interface of PCNA. The new mode of Rev1-PCNA binding described here suggests a mechanism by which Rev1 adopts a catalytically inactive configuration at the replication fork.     

Volatile secondary metabolites of Micromeria dalmatica Benth. (Lamiaceae): biosynthetical and chemotaxonomical aspects

Analysis by GC and GC/MS of the essential oil obtained from above-ground parts of Micromeria dalmatica Benth. allowed the identification of 116 components, comprising 93.6% of the total oil composition. The major compounds are 3-oxygenated p-menthane monoterpenes and were identified as pulegone (29.6%), menthone (11.7%), and piperitenone (10.8%). The chemical composition of this and additional 30 oils obtained from selected Micromeria Benth. taxa were compared by using multivariate statistical analysis (agglomerative hierarchical cluster analysis and principal component analysis (PCA)). The results of statistical analyses, as well as the domination of different concurrent p-menthane-skeleton-type monoterpene biosynthetical sub-branches in the compared M. dalmatica samples, implied the occurrence of at least two different chemotypes of the mentioned species.     

Spatial-Orientation Priming Impedes Rather than Facilitates the Spontaneous Control of Hand-Retraction Speeds in Patients with Parkinson’s Disease

Background

Often in Parkinson’s disease (PD) motor-related problems overshadow latent non-motor deficits as it is difficult to dissociate one from the other with commonly used observational inventories. Here we ask if the variability patterns of hand speed and acceleration would be revealing of deficits in spatial-orientation related decisions as patients performed a familiar reach-to-grasp task. To this end we use spatial-orientation priming which normally facilitates motor-program selection and asked whether in PD spatial-orientation priming helps or hinders performance.

Methods

To dissociate spatial-orientation- and motor-related deficits participants performed two versions of the task. The biomechanical version (DEFAULT) required the same postural- and hand-paths as the orientation-priming version (primed-UP). Any differences in the patients here could not be due to motor issues as the tasks were biomechanically identical. The other priming version (primed-DOWN) however required additional spatial and postural processing. We assessed in all three cases both the forward segment deliberately aimed towards the spatial-target and the retracting segment, spontaneously bringing the hand to rest without an instructed goal.

Results and Conclusions

We found that forward and retracting segments belonged in two different statistical classes according to the fluctuations of speed and acceleration maxima. Further inspection revealed conservation of the forward (voluntary) control of speed but in PD a discontinuity of this control emerged during the uninstructed retractions which was absent in NC. Two PD groups self-emerged: one group in which priming always affected the retractions and the other in which only the more challenging primed-DOWN condition was affected. These PD-groups self-formed according to the speed variability patterns, which systematically changed along a gradient that depended on the priming, thus dissociating motor from spatial-orientation issues. Priming did not facilitate the motor task in PD but it did reveal a breakdown in the spatial-orientation decision that was independent of the motor-postural path.     

Structural analyses of the ankyrin repeat domain of TRPV6 and related TRPV ion channels

Transient receptor potential (TRP) proteins are cation channels composed of a transmembrane domain flanked by large N- and C-terminal cytoplasmic domains. All members of the vanilloid family of TRP channels (TRPV) possess an N-terminal ankyrin repeat domain (ARD). The ARD of mammalian TRPV6, an important regulator of calcium uptake and homeostasis, is essential for channel assembly and regulation. The 1.7 A crystal structure of the TRPV6-ARD reveals conserved structural elements unique to the ARDs of TRPV proteins. First, a large twist between the fourth and fifth repeats is induced by residues conserved in all TRPV ARDs. Second, the third finger loop is the most variable region in sequence, length and conformation. In TRPV6, a number of putative regulatory phosphorylation sites map to the base of this third finger. Size exclusion chromatography and crystal packing indicate that the TRPV6-ARD does not assemble as a tetramer and is monomeric in solution. Adenosine triphosphate-agarose and calmodulin-agarose pull-down assays show that the TRPV6-ARD does not interact with either ligand, indicating a different functional role for the TRPV6-ARD than in the paralogous thermosensitive TRPV1 channel. Similar biochemical findings are also presented for the highly homologous mammalian TRPV5-ARD. The implications of the structural and biochemical data on the role of the ankyrin repeats in different TRPV channels are discussed.