135 research outputs found
Kinetic and pH Studies on Human Phenylethanolamine N-Methyltransferase
Phenylethanolamine N-methyltransferase (PNMT) catalyzes the conversion of norepinephrine (noradrenaline) to epinephrine (adrenaline) while, concomitantly, S-adenosyl-l-methionine (AdoMet) is converted to S-adenosyl-l-homocysteine. This reaction represents the terminal step in catecholamine biosynthesis and inhibitors of PNMT have been investigated, inter alia, as potential antihypertensive agents. At various times the kinetic mechanism of PNMT has been reported to operate by a random mechanism, an ordered mechanism in which norepinephrine binds first, and an ordered mechanism in which AdoMet binds first. Here we report the results of initial velocity studies on human PNMT in the absence and presence of product and dead end inhibitors. These, coupled with isothermal titration calorimetry and fluorescence binding experiments, clearly shown that hPNMT operates by an ordered sequential mechanism in which AdoMet binds first. Although the log V pH-profile was not well defined, plots of log V/K versus pH for AdoMet and phenylethanolamine, as well as the pKi versus pH for the inhibitor, SK&F 29661, were all bell-shaped indicating that a protonated and an unprotonated group are required for catalysis
Codivergence and multiple host species use by fig wasp populations of the Ficus pollination mutualism
<p>Abstract</p> <p>Background</p> <p>The interaction between insects and plants takes myriad forms in the generation of spectacular diversity. In this association a species host range is fundamental and often measured using an estimate of phylogenetic concordance between species. Pollinating fig wasps display extreme host species specificity, but the intraspecific variation in empirical accounts of host affiliation has previously been underestimated. In this investigation, lineage delimitation and codiversification tests are used to generate and discuss hypotheses elucidating on pollinating fig wasp associations with <it>Ficus</it>.</p> <p>Results</p> <p>Statistical parsimony and AMOVA revealed deep divergences at the <it>COI </it>locus within several pollinating fig wasp species that persist on the same host <it>Ficus </it>species. Changes in branching patterns estimated using the generalized mixed Yule coalescent test indicated lineage duplication on the same <it>Ficus </it>species. Conversely, <it>Elisabethiella </it>and <it>Alfonsiella </it>fig wasp species are able to reproduce on multiple, but closely related host fig species. Tree reconciliation tests indicate significant codiversification as well as significant incongruence between fig wasp and <it>Ficus </it>phylogenies.</p> <p>Conclusions</p> <p>The findings demonstrate more relaxed pollinating fig wasp host specificity than previously appreciated. Evolutionarily conservative host associations have been tempered by horizontal transfer and lineage duplication among closely related <it>Ficus </it>species. Independent and asynchronistic diversification of pollinating fig wasps is best explained by a combination of both sympatric and allopatric models of speciation. Pollinator host preference constraints permit reproduction on closely related <it>Ficus </it>species, but uncertainty of the frequency and duration of these associations requires better resolution.</p
Host-driven diversification of gall-inducing Acacia thrips and the aridification of Australia
BACKGROUND: Insects that feed on plants contribute greatly to the generation of biodiversity. Hypotheses explaining rate increases in phytophagous insect diversification and mechanisms driving speciation in such specialists remain vexing despite considerable attention. The proliferation of plant-feeding insects and their hosts are expected to broadly parallel one another where climate change over geological timescales imposes consequences for the diversification of flora and fauna via habitat modification. This work uses a phylogenetic approach to investigate the premise that the aridification of Australia, and subsequent expansion and modification of arid-adapted host flora, has implications for the diversification of insects that specialise on them. RESULTS: Likelihood ratio tests indicated the possibility of hard molecular polytomies within two co-radiating gall-inducing species complexes specialising on the same set of host species. Significant tree asymmetry is indicated at a branch adjacent to an inferred transition to a Plurinerves ancestral host species. Lineage by time diversification plots indicate gall-thrips that specialise on Plurinerves hosts differentially experienced an explosive period of speciation contemporaneous with climatic cycling during the Quaternary period. Chronological analyses indicated that the approximate age of origin of gall-inducing thrips on Acacia might be as recent as 10 million years ago during the Miocene, as truly arid landscapes first developed in Australia. CONCLUSION: Host-plant diversification and spatial heterogeneity of hosts have increased the potential for specialisation, resource partitioning, and unoccupied ecological niche availability for gall-thrips on Australian Acacia
Computational Study of Enantioselective Carboligation Catalyzed by Benzoylformate Decarboxylase
Benzoylformate decarboxylase (BFDC) is a thiamin-diphosphate enzyme that catalyzes the decarboxylation of benzoylformate to yield benzaldehyde and carbon dioxide. In addition to its natural reaction, BFDC is able to catalyze carboligation reactions in a highly enantioselective fashion, making the enzyme a potentially important biocatalyst. Here we use density functional theory calculations to investigate the detailed mechanism of BFDC-catalyzed carboligation and to elucidate the sources of the enantioselectivity. Benzaldehyde and acetaldehyde are studied as acceptors, for, when reacting with a benzaldehyde donor, they yield products with opposite enantiospecificity. For each of the acceptors, several possible binding modes to the active site are initially examined before the individual reaction paths leading to the two enantiomeric products are followed. The calculated energies are in good agreement with the experimental results, and the analysis of the transition states gives insight into the origins of the enantioselectivity
Using saturation mutagenesis to explore substrate specificity and catalysis in benzoylformate decarboxylase
poster abstractBenzoylformate decarboxylase (BFDC) from Pseudomonas putida and pyruvate decarboxylase (PDC) from Zymomonas mobilis are thiamin diphosphate (ThDP)-dependent enzymes. The two share a common three-dimensional structure and catalyze a similar chemical reaction, i.e., decarboxylation of 2-keto acids. However, they vary significantly in their substrate utilization pattern. In particular, BFDC has extremely limited activity with pyruvate, while PDC has no activity with benzoylformate. Both enzymes also catalyze stereospecific carboligation reactions that are of commercial interest, again with a different range of substrates. In order to identify similarities and differences on a molecular level, and to reveal factors responsible for substrate specificity and enantioselectivity, the X-ray structures BFDC and PDC were compared. Residues identified in this process were subjected to site-directed mutagenesis. The results show that, although it was not possible to simply interchange substrates, it was possible to engineer enzymes that had distinctly different substrate specificities while retaining excellent kinetic activity. However, it also became apparent that a more general approach was needed. Towards this end we developed a screening procedure for BFDC to enable us to use saturation mutagenesis to examine residues involved in substrate specificity. During the development of the methodology it became clear that it was possible to use this approach to explore residues involved in catalysis by BFDC. Here we describe the unexpected results obtained using saturation mutagenesis on putative catalytic residues. In addition we report towards converting BFDC into an efficient pyruvate decarboxylase
Rates and Intermediates in Ser26 Mutants of Benzoylformate Decarboxylase
poster abstractBenzoylformate decarboxylase (BFDC), a thiamine diphosphate dependent enzyme,
catalyzes decarboxylation of benzoylformate to benzaldehyde and CO2. The BFDC
reaction proceeds through at least four individual chemical steps and, recently, NMR
spectroscopy has been used to measure the ratios of intermediates in the overall reaction.
This method permits calculation of rate constants for formation of the first intermediate,
mandelylThDP (k2) and its subsequent decarboxylation (k3), as well as the combined
breakdown of the enamine and product release (k4). As part of a study of the
contributions of the active site residues, Ser26, His70 and His281, to the individual
catalytic steps several Ser26 variants were expressed and purified. Initially, the variants
were characterized using steady-state kinetics. Subsequently, the enzymes were mixed
with benzoylformate and the mixture immediately acid quenched to trap intermediates of
the reaction. NMR spectroscopy was used to identify and quantitate individual catalytic
intermediates. Rate constants for the formation of these intermediates were then
determined and compared to those of the wild-type enzyme. Here we report those results
and discuss their implications for the role of Ser26 in the BFDC reaction mechanism
A Theoretical Study of the Benzoylformate Decarboxylase Reaction Mechanism
Density functional theory calculations are used to investigate the detailed reaction mechanism of benzoylformate decarboxylase, a thiamin diphosphate (ThDP)-dependent enzyme that catalyzes the nonoxidative decarboxylation of benzoylformate yielding benzaldehyde and carbon dioxide. A large model of the active site is constructed on the basis of the X-ray structure, and it is used to characterize the involved intermediates and transition states and evaluate their energies. There is generally good agreement between the calculations and available experimental data. The roles of the various active site residues are discussed and the results are compared to mutagenesis experiments. Importantly, the calculations identify off-cycle intermediate species of the ThDP cofactor that can have implications on the kinetics of the reaction
Phosphonodifluoropyruvate is a mechanism-based inhibitor of phosphonopyruvate decarboxylase from Bacteroides fragilis
Bacteroides fragilis, a human pathogen, helps in the formation of intra-abdominal abscesses and is involved in 90% of anaerobic peritoneal infections. Phosphonopyruvate decarboxylase (PnPDC), a thiamin diphosphate (ThDP)-dependent enzyme, plays a key role in the formation of 2-aminoethylphosphonate, a component of the cell wall of B. fragilis. As such PnPDC is a possible target for therapeutic intervention in this, and other phosphonate producing organisms. However, the enzyme is of more general interest as it appears to be an evolutionary forerunner to the decarboxylase family of ThDP-dependent enzymes. To date, PnPDC has proved difficult to crystallize and no X-ray structures are available. In the past we have shown that ThDP-dependent enzymes will often crystallize if the cofactor has been irreversibly inactivated. To explore this possibility, and the utility of inhibitors of phosphonate biosynthesis as potential antibiotics, we synthesized phosphonodifluoropyruvate (PnDFP) as a prospective mechanism-based inhibitor of PnPDC. Here we provide evidence that PnDFP indeed inactivates the enzyme, that the inactivation is irreversible, and is accompanied by release of fluoride ion, i.e., PnDFP bears all the hallmarks of a mechanism-based inhibitor. Unfortunately, the enzyme remains refractive to crystallization
Specificity and Mechanism of Mandelamide Hydrolase Catalysis
The best-studied amidase signature (AS) enzyme is probably fatty acid amide hydrolase (FAAH). Closely related to FAAH is mandelamide hydrolase (MAH), whose substrate specificity and mechanism of catalysis are described in this paper. First, we developed a convenient chromogenic substrate, 4-nitrophenylacetamide, for MAH. The lack of reactivity of MAH with the corresponding ethyl ester confirmed the very limited size of the MAH leaving group site. The reactivity of MAH with 4-nitrophenyl acetate and methyl 4-nitrophenyl carbonate, therefore, suggested formation of an βinverseβ acyl-enzyme where the small acyl-group occupies the normal leaving group site. We have interpreted the specificity of MAH for phenylacetamide substrates and small leaving groups in terms of its active site structure, using a homology model based on a FAAH crystal structure. The relevant structural elements were compared with those of FAAH. Phenylmethylboronic acid is a potent inhibitor of MAH (Ki = 27 nM), presumably because it forms a transition state analogue structure with the enzyme. O-Acyl hydroxamates were not irreversible inactivators of MAH but some were found to be transient inhibitors
Host-plant species conservatism and ecology of a parasitoid fig wasp genus (Chalcidoidea; Sycoryctinae; Arachonia)
Parasitoid diversity in terrestrial ecosystems is enormous. However, ecological processes underpinning their evolutionary diversification in association with other trophic groups are still unclear. Specialisation and interdependencies among chalcid wasps that reproduce on Ficus presents an opportunity to investigate the ecology of a multi-trophic system that includes parasitoids. Here we estimate the host-plant species specificity of a parasitoid fig wasp genus that attacks the galls of non-pollinating pteromalid and pollinating agaonid fig wasps. We discuss the interactions between parasitoids and the Ficus species present in a forest patch of Uganda in context with populations in Southern Africa. Haplotype networks are inferred to examine intraspecific mitochondrial DNA divergences and phylogenetic approaches used to infer putative species relationships. Taxonomic appraisal and putative species delimitation by molecular and morphological techniques are compared. Results demonstrate that a parasitoid fig wasp population is able to reproduce on at least four Ficus species present in a patch. This suggests that parasitoid fig wasps have relatively broad host- Ficus species ranges compared to fig wasps that oviposit internally. Parasitoid fig wasps did not recruit on all available host plants present in the forest census area and suggests an important ecological consequence in mitigating fitness trade-offs between pollinator and Ficus reproduction. The extent to which parasitoid fig wasps exert influence on the pollination mutualism must consider the fitness consequences imposed by the ability to interact with phenotypes of multiple Ficus and fig wasps species, but not equally across space and time
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