Novel endo-α-N-acetylgalactosaminidases with broader substrate specificity

In an effort to identify novel endo-α-N-acetylgalact- osaminidases (endo-α-GalNAcases), four potential genes were cloned. Three of the expressed proteins EngEF from Enterococcus faecalis, EngPA from Propionibacterium acnes, and EngCP from Clostridium perfringens were purified and characterized. Their substrate specificity was investigated and compared to the commercially available endo-α-GalNAcases from Streptococcus pneumoniae (EngSP) and Alcaligenes sp. (EngAL). All enzymes were incubated with various synthetic substrates, and natural glycoproteins and the released sugars were detected by colorimetric assay and thin layer chromatography analysis. The Core 1 disaccharide Galβ1,3GalNAcα1pNP was the most rapidly hydrolyzed substrate by all enzymes tested. EngEF exhibited the highest kcat for this substrate. EngEF and EngPA were also able to fully hydrolyze the Core 3 disaccharide GlcNAcβ1,3GalNAcα1pNP. This is the first report of endo-α-GalNAcases EngEF and EngPA acting on Core 3 in addition to Core 1 O-glycans. Interestingly, there were no significant differences in transglycosylation activities when Galβ1,3GalNAcα1pNP or GlcNAcβ1,3GalNAcα1pNP was incubated with various 1-alkanols in the presence of the endo-α-GalNAcases tested in this work

Carbohydrate Recognition by an Architecturally Complex α-N-Acetylglucosaminidase from Clostridium perfringens

CpGH89 is a large multimodular enzyme produced by the human and animal pathogen Clostridium perfringens. The catalytic activity of this exo-α-d-N-acetylglucosaminidase is directed towards a rare carbohydrate motif, N-acetyl-β-d-glucosamine-α-1,4-d-galactose, which is displayed on the class III mucins deep within the gastric mucosa. In addition to the family 89 glycoside hydrolase catalytic module this enzyme has six modules that share sequence similarity to the family 32 carbohydrate-binding modules (CBM32s), suggesting the enzyme has considerable capacity to adhere to carbohydrates. Here we suggest that two of the modules, CBM32-1 and CBM32-6, are not functional as carbohydrate-binding modules (CBMs) and demonstrate that three of the CBMs, CBM32-3, CBM32-4, and CBM32-5, are indeed capable of binding carbohydrates. CBM32-3 and CBM32-4 have a novel binding specificity for N-acetyl-β-d-glucosamine-α-1,4-d-galactose, which thus complements the specificity of the catalytic module. The X-ray crystal structure of CBM32-4 in complex with this disaccharide reveals a mode of recognition that is based primarily on accommodation of the unique bent shape of this sugar. In contrast, as revealed by a series of X-ray crystal structures and quantitative binding studies, CBM32-5 displays the structural and functional features of galactose binding that is commonly associated with CBM family 32. The functional CBM32s that CpGH89 contains suggest the possibility for multivalent binding events and the partitioning of this enzyme to highly specific regions within the gastrointestinal tract

Κατευθυνόμενη εξέλιξη δύο ενζυμικών μορίων

In the present study we employed directed evolution in order to improve selected properties of two enzymes: chitin deacetylase Cda2p from the zymomyces Saccharomyces cerevisiae and alkaline phosphatase from the Antarctic strain TAB5. The properties-targets for Cda2p were product inhibition by acetic acid and the incapability of the enzyme to act upon insoluble chitin substrates, while for TAB5AP the low thermostability. We applied error prone PCR on both genes and subsequently screened the mutated libraries. Unfortunately, the existing screening assays for Cda2p, didn't match with the needs of our experiments, so we were unable to screen the mutated clones. On the other hand we screened approximately 5500 clones from the TAB5AP libraries and selected 12 clones, which demonstrated higher levels of thermostability as compared to the wild type enzyme. Finally, we cloned and overexpressed the CDA2 gene in Escherichia coli. At the same time we designed two schemes of partial purification of the recombinant protein.Στην παρούσα εργασία χρησιμοποιήθηκε η τεχνική της κατευθυνόμενης εξέλιξης για την τροποποίηση ενζυμικών ιδιοτήτων. Δυο ένζυμα αποτέλεσαν τo αντικείμενo της ερευνητικής προσπάθειας: η απακετυλάση της χιτίνης Cda2p από το ζυμομύκητα Saccharomyces cerevisiae και η αλκαλική φωσφατάση από το ανταρκτικό στέλεχος ΤΑΒ5. Οι ιδιότητες στόχοι για την Cda2p ήταν η αναστολή του ενζύμου από το οξικό οξύ και η αδυναμία δράσης σε αδιάλυτα υποστρώματα χιτίνης, ενώ για την ΤΑΒ5ΑΡ ήταν η χαμηλή θερμοσταθερότητα. Μετά από τη χρήση της τεχνικής error prone PCR στα παραπάνω γονίδια, ακολούθησε η σάρωση των βιβλιοθηκών από μεταλλαγμένους κλώνους. Στην περίπτωση της Cda2p και λόγω της ακαταλληλότητας των διαθέσιμων τεχνικών σάρωσης, δεν έγινε δυνατός ο έλεγχος των μεταλλαγμένων κλώνων. Στην περίπτωση όμως της ΤΑΒ5ΑΡ ελέγχθηκαν περίπου 5500 κλώνοι από τους οποίους 12 εμφανίστηκαν με υψηλότερη θερμοσταθερότητα από το ένζυμο αγρίου τύπου. Τέλος στην προσπάθεια έκφρασης του ενζύμου Cda2p σε ικανοποιητικά επίπεδα κλωνοποιήθηκε το αντίστοιχο γονίδιο στο βακτήριο Escherichia coli. Παράλληλα σχεδιάστηκαν δύο επιτυχή σχήματα μερικής απομόνωσης της ανασυνδυασμένης πρωτεΐνης

Directed evolution on the cold adapted properties of TAB5 alkaline phosphatase

Psychrophilic alkaline phosphatase (AP) from the Antarctic strain TAB5 was subjected to directed evolution in order to identify the key residues steering the enzyme's cold-adapted activity and stability. A round of random mutagenesis and further recombination yielded three thermostable and six thermolabile variants of the TAB5 AP. All of the isolated variants were characterised by their residual activity after heat treatment, Michaelis-Menten kinetics, activation energy and microcalorimetric parameters of unfolding. In addition, they were modelled into the structure of the TAB5 AP. Mutations which affected the cold-adapted properties of the enzyme were all located close to the active site. The destabilised variants H135E and H135E/G149D had 2- and 3-fold higher kcat, respectively, than the wild-type enzyme. Wild-type AP has a complex heat-induced unfolding pattern while the mutated enzymes loose local unfolding transitions and have large shifts of the Tm values. Comparison of the wild-type and mutated TAB5 APs demonstrates that there is a delicate balance between the enzyme activity and stability and that it is possible to improve the activity and thermostability simultaneously as demonstrated in the case of the H135E/G149D variant compared to H135E

Structure determination of BA0150, a putative polysaccharide deacetylase from Bacillus anthracis

Polysaccharide deacetylases are bacterial enzymes that catalyze the deacetylation of acetylated sugars on the membranes of Gram-positive bacteria, allowing them to be unrecognized by host immune systems. Inhibition of these enzymes would disrupt such pathogenic defensive mechanisms and therefore offers a promising route for the development of novel antibiotic therapeutics. Here, the first X-ray crystal structure of BA0150, a putative polysaccharide deacetylase from Bacillus anthracis, is reported to 2.0Å resolution. The overall structure maintains the conserved (α/β)8 fold that is characteristic of this family of enzymes. The lack of a catalytic metal ion and a distinctive metal-binding site, however, suggest that this enzyme is not a functional polysaccharide deacetylase. © 2014 International Union of Crystallography All rights reserved