The three-dimensional structure of an unusually active hydrolytic antibody with a phosphonate transition state analog (hapten) bound to the active site has been solved to 2.5 A resolution. The antibody (17E8) catalyzes the hydrolysis of norleucine and methionine phenyl esters and is selective for amino acid esters that have the natural alpha-carbon L configuration. A plot of the pH-dependence of the antibody-catalyzed reaction is bell-shaped with an activity maximum at pH 9.5; experiments on mechanism lend support to the formation of a covalent acyl-antibody intermediate. The structural and kinetic data are complementary and support a hydrolytic mechanism for the antibody that is remarkably similar to that of the serine proteases. The antibody active site contains a Ser-His dyad structure proximal to the phosphorous atom of the bound hapten that resembles two of the three components of the Ser-His-Asp catalytic triad of serine proteases. The antibody active site also contains a Lys residue to stabilize oxyanion formation, and a hydrophobic binding pocket for specific substrate recognition of norleucine and methionine side chains. The structure identifies active site residues that mediate catalysis and suggests specific mutations that may improve the catalytic efficiency of the antibody. This high resolution structure of a catalytic antibody-hapten complex shows that antibodies can converge on active site structures that have arisen through natural enzyme evolution.
Several steps of the reaction catalyzed by thymidylate synthase (TS) require proton transfers to and from O-4 and C-5 of the pyrimidine moiety of substrate dUMP. It has been proposed that one or more of three active site residues-Glu60, His199, and Asn229-together with ordered water molecules serve as general catalysts in facilitating such proton transfers. These three residues, individually and together were mutated to residues incapable of proton transfer, and the mutant enzymes were purified and tested for activity in the formation of dTMP and the dehalogenation of 5-bromo- and 5-iodo-dUMP. The dehalogenation reaction pathway shares at least two direct chemical counterparts with the TS reaction pathway which are believed to involve general acid/base catalysis-namely, the addition and elimination of the catalytic Cys of TS at C-6 of the pyrimidine substrate. Generally, the mutations had detrimental effects on dTMP synthesis with the triple mutant being completely inactive. In contrast, single mutants TS E601, and H199A and, interestingly, the triple mutant stripped of all three active site catalysts catalyzed the dehalogenation reaction as well as or better than the wild-type enzyme. It was concluded that addition and elimination reactions involving the 5.6-bond of pyrimidine substrates do not require general acid/base catalysis or, alternatively, the water molecules in the TS active site serve this role. The function(s) of the triad of general catalysts resides elsewhere in the reaction pathway leading to dTMP synthesis.
Even though ladybirds are well known as economically important biological control agents, an integrative framework of DNA barcoding research was not available for the family so far. We designed and present a set of efficient mini-barcoding primers to recover full DNA barcoding sequences for Coccinellidae, even for specimens collected 40 years ago. Based on these mini-barcoding primers, we obtained 104 full DNA barcode sequences for 104 species of Coccinellidae, in which 101 barcodes were newly reported for the first time. We also downloaded 870 COI barcode sequences (658 bp) from GenBank and BOLD database, belonging to 108 species within 46 genera, to assess the optimum genetic distance threshold and compare four methods of species delimitation (GMYC, bPTP, BIN and ABGD) to determine the most accurate approach for the family. The results suggested the existence of a 'barcode gap' and that 3% is likely an appropriate genetic distance threshold to delimit species of Coccinellidae using DNA barcodes. Species delimitation analyses confirm ABGD as an accurate and efficient approach, more suitable than the other three methods. Our research provides an integrative framework for DNA barcoding and descriptions of new taxa in Coccinellidae. Our results enrich DNA barcoding public reference libraries, including data for Chinese coccinellids. This will facilitate taxonomic identification and biodiversity monitoring of ladybirds using metabarcoding. As species are a fundamental biological category, accurately identifying them is an essential premise of biological studies. Tautz et al. 1 proposed DNA sequences as a species identification system for the first time. Subsequently, the 5' end of the mitochondrial cytochrome c oxidase subunit I gene (COI) has been suggested as a standardized DNA "barcode" for identifying species of all groups of animals, launching the DNA barcoding technology 2. The success of barcode identification based upon genetic distances ultimately depended on differences between intraand interspecific divergences 2-4. DNA barcoding using the 658 bp 5' region of mtCOI DNA sequence as a tool, has turned out as very efficient and reliable for identifying specimens of unknown origin and taxonomic status, and also for the identification of different developmental stages. The approximately 1600 base-pairs comprise a range of different functional domains showing heterogenous substitution patterns 5,6. In addition to its utility for distinguishing known species, the COI region has also been found suitable for revealing cryptic species 7-11 , and biogeographic and phylogeographic patterns, and also species level phylogenetic relationships 12-14. The family Coccinellidae is placed in the superfamily Coccinelloidea within the Coleopteran suborder Polyphaga 15,16. Coccinellids are well known as economically important biological control agents, but this family is ecologically and morphologically very diverse. It comprises about 490 genera and nearly 6000 described species worldwide 17. Due to relatively small body size ...
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