Quantitative Comparison of Catalytic Mechanisms and Overall Reactions in Convergently Evolved Enzymes: Implications for Classification of Enzyme Function

Functionally analogous enzymes are those that catalyze similar reactions on similar substrates but do not share common ancestry, providing a window on the different structural strategies nature has used to evolve required catalysts. Identification and use of this information to improve reaction classification and computational annotation of enzymes newly discovered in the genome projects would benefit from systematic determination of reaction similarities. Here, we quantified similarity in bond changes for overall reactions and catalytic mechanisms for 95 pairs of functionally analogous enzymes (non-homologous enzymes with identical first three numbers of their EC codes) from the MACiE database. Similarity of overall reactions was computed by comparing the sets of bond changes in the transformations from substrates to products. For similarity of mechanisms, sets of bond changes occurring in each mechanistic step were compared; these similarities were then used to guide global and local alignments of mechanistic steps. Using this metric, only 44% of pairs of functionally analogous enzymes in the dataset had significantly similar overall reactions. For these enzymes, convergence to the same mechanism occurred in 33% of cases, with most pairs having at least one identical mechanistic step. Using our metric, overall reaction similarity serves as an upper bound for mechanistic similarity in functional analogs. For example, the four carbon-oxygen lyases acting on phosphates (EC 4.2.3) show neither significant overall reaction similarity nor significant mechanistic similarity. By contrast, the three carboxylic-ester hydrolases (EC 3.1.1) catalyze overall reactions with identical bond changes and have converged to almost identical mechanisms. The large proportion of enzyme pairs that do not show significant overall reaction similarity (56%) suggests that at least for the functionally analogous enzymes studied here, more stringent criteria could be used to refine definitions of EC sub-subclasses for improved discrimination in their classification of enzyme reactions. The results also indicate that mechanistic convergence of reaction steps is widespread, suggesting that quantitative measurement of mechanistic similarity can inform approaches for functional annotation.     

Insulin Degrading Enzyme Induces a Conformational Change in Varicella-Zoster Virus gE,and Enhances Virus Infectivity and Stability

Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for virus infectivity and binds to a cellular receptor, insulin-degrading enzyme (IDE), through its unique amino terminal extracellular domain. Previous work has shown IDE plays an important role in VZV infection and virus cell-to-cell spread, which is the sole route for VZV spread in vitro. Here we report that a recombinant soluble IDE (rIDE) enhances VZV infectivity at an early step of infection associated with an increase in virus internalization, and increases cell-to-cell spread. VZV mutants lacking the IDE binding domain of gE were impaired for syncytia formation and membrane fusion. Pre-treatment of cell-free VZV with rIDE markedly enhanced the stability of the virus over a range of conditions. rIDE interacted with gE to elicit a conformational change in gE and rendered it more susceptible to proteolysis. Co-incubation of rIDE with gE modified the size of gE. We propose that the conformational change in gE elicited by IDE enhances infectivity and stability of the virus and leads to increased fusogenicity during VZV infection. The ability of rIDE to enhance infectivity of cell-free VZV over a wide range of incubation times and temperatures suggests that rIDE may be useful for increasing the stability of varicella or zoster vaccines.     

Phylogeny of magpie‐robins and shamas (Aves: Turdidae: Copsychus and Trichixos): implications for island biogeography in Southeast Asia

Aim Magpie‐robins and shamas are forest and woodland birds of south Asia. There are two genera: Trichixos for the monotypic T. pyrrhopygus, and Copsychus for other species. Two species are widespread, whereas the others are restricted to specific islands. Endemicity is highest in the Philippines. Using phylogenetic methods, we examined how this group came to its unusual distribution. Location Mainland Asia from India to southern China, and islands from Madagascar to the Philippines. Particular emphasis is placed on the Greater Sundas and Philippines. Methods The phylogeny was estimated from DNA sequences of 14 ingroup taxa representing all nine currently recognized Copsychus and Trichixos species. The entire mitochondrial ND2 gene and portions of nuclear myoglobin intron 2 (Myo2) and transforming growth factor beta 2 intron 5 (TGFβ2‐5) were sequenced for all but two species. The phylogeny was reconstructed using maximum likelihood and Bayesian methods. The timing of divergence events was estimated using a relaxed molecular clock approach, and ancestral areas were examined using stochastic modelling. Results The group comprises three main clades corresponding to ecological types: Trichixos, a primary‐forest specialist; Copsychus magpie‐robins, open‐woodland and coastal species; and Copsychus shamas, thick‐forest species. Trichixos appears to be sister to the magpie‐robins, rendering Copsychus polyphyletic. The dating of phylogenetic nodes was too ambiguous to provide substantial insight into specific geographical events responsible for divergence within the group. Some patterns are nevertheless clear. Copsychus shamas reached the Philippines, probably in two separate invasions, and split into endemic species. Copsychus malabaricus and C. saularis expanded widely in the Greater Sundas and mainland Southeast Asia without species‐level diversification. Main conclusions Magpie‐robins are excellent dispersers and have diversified into distinct species only on isolated oceanic islands. Trichixos, a poor disperser, is restricted to mature forests of the Malay Peninsula, Sumatra and Borneo. Copsychus shamas are intermediate in habitat preference and dispersal capabilities. Their endemism in the Philippines may be attributed to early colonization and specialization to interior forests. In the Greater Sundas, C. malabaricus and C. saularis populations split and came together on Borneo to form two separate subspecies (of each species), which now hybridize.     

Estimating nests not present at the time of breeding surveys: an important consideration in assessing nesting populations

ABSTRACT.   Counts of nest starts are often used as indicators of the size of avian nesting populations, or of avian productivity. However, the accuracy of single or repeated counts of unmarked nests over time for estimating seasonal numbers of nests may be strongly affected by nest events that fall in between survey dates, or that occurred prior to or after the survey period. Accuracy may also be affected by uncertainty in the interpretation of counts due to overlap between starting and ending dates of asynchronous nests during the intervisit interval. To measure the combined magnitude of these effects on survey accuracy, we overlaid a monthly "survey" regime on known initiation and ending dates of 2055 nests of ciconiiform birds. Assuming all nests present on the date of simulated survey were counted, monthly surveys underestimated the true number of nest starts by 24–64%, depending on species and year. Using a simple model, we also demonstrate that accuracy does not increase much as survey frequency increases, and that significant estimation error can occur over a wide range of nest success values and degrees of asynchrony. We suggest that (1) these biases can be significant for surveys of many kinds of nesting birds including some territorial passerines, (2) this bias cannot be addressed by increasing survey frequency, and (3) the degree of renesting may be of critical interest for inferring breeding population size from nest count data. We suggest three possible approaches for modeling this error.     

A fight for neurotransmission: SCRAPPER trashes RIM

The presynaptic scaffold molecule RIM1alpha is important for regulating neurotransmitter release. In this issue, Yao et al. (2007) show in mice that an E3 ubiquitin ligase, SCRAPPER, targets a set of presynaptic proteins including RIM1alpha for degradation by the ubiquitin-proteasome system. Their results identify protein degradation as a mechanism for holding rapid synaptic communication in check.     

Sensitivity and specificity of culture and PCR of smegma samples of bulls experimentally infected with Tritrichomonas foetus

The sensitivity (Se) and specificity (Sp) of different testing schemes were estimated for detecting Tritrichomonas foetus (T. foetus) in smegma samples from experimentally infected bulls. Culture and polymerase chain reaction (PCR) on smegma samples were evaluated alone and in parallel testing. Mature dairy bulls (n=79) were intrapreputially inoculated with T. foetus (n=19); Campylobacter (C.) fetus venerealis (n=13); both T. foetus and C. fetus venerealis (n=11); Tetratrichomonas spp. (n=9); C. fetus fetus (n=8); or were not inoculated (n=19). For each bull, smegma samples were collected for 6 week post-inoculation and tested for T. foetus by In Pouch TF culture and PCR. Most T. foetus-inoculated bulls became infected, according to culture (86.7%), PCR (90.0%), and both tests together (93.3%). In T. foetus-inoculated bulls, both tests combined in parallel on a single sample had a Se (78.3%) and Sp (98.5%) similar to two cultures (Se 76.0%, Sp 98.5%) or two PCR (Se 78.0%, Sp 96.7%) sampled on consecutive weeks. The PCR on three consecutive weekly samples (Se 85.0%, Sp 95.4%) and both tests applied in parallel on three consecutive weekly samples (Se 87.5%, Sp 95.6%) were similar to the current gold-standard of six weekly cultures (Se 86.7% and Sp 97.5%). Both tests used in parallel six times had the highest Se (93.3%), with similar Sp (92.5%). Tetratrichomonas spp. were only sporadically detected by culture or PCR. In conclusion, we have proposed alternative strategies for T. foetus diagnostics (for the AI industry), including a combination of tests and repeat testing strategies that may reduce time and cost for bull surveillance.