The evolution of cyclodextrin glucanotransferase product specificity

Cyclodextrin glucanotransferases (CGTases) have attracted major interest from industry due to their unique capacity of forming large quantities of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. CGTases produce a mixture of cyclodextrins from starch consisting of 6 (α), 7 (β) and 8 (γ) glucose units. In an effort to identify the structural factors contributing to the evolutionary diversification of product specificity amongst this group of enzymes, we selected nine CGTases from both mesophilic, thermophilic and hyperthermophilic organisms for comparative product analysis. These enzymes displayed considerable variation regarding thermostability, initial rates, percentage of substrate conversion and ratio of α-, β- and γ-cyclodextrins formed from starch. Sequence comparison of these CGTases revealed that specific incorporation and/or substitution of amino acids at the substrate binding sites, during the evolutionary progression of these enzymes, resulted in diversification of cyclodextrin product specificity

UPF201 Archaeal Specific Family Members Reveal Structural Similarity to RNA-Binding Proteins but Low Likelihood for RNA-Binding Function

We have determined X-ray crystal structures of four members of an archaeal specific family of proteins of unknown function (UPF0201; Pfam classification: DUF54) to advance our understanding of the genetic repertoire of archaea. Despite low pairwise amino acid sequence identities (10–40%) and the absence of conserved sequence motifs, the three-dimensional structures of these proteins are remarkably similar to one another. Their common polypeptide chain fold, encompassing a five-stranded antiparallel β-sheet and five α-helices, proved to be quite unexpectedly similar to that of the RRM-type RNA-binding domain of the ribosomal L5 protein, which is responsible for binding the 5S- rRNA. Structure-based sequence alignments enabled construction of a phylogenetic tree relating UPF0201 family members to L5 ribosomal proteins and other structurally similar RNA binding proteins, thereby expanding our understanding of the evolutionary purview of the RRM superfamily. Analyses of the surfaces of these newly determined UPF0201 structures suggest that they probably do not function as RNA binding proteins, and that this domain specific family of proteins has acquired a novel function in archaebacteria, which awaits experimental elucidation

Fungal chitinases: diversity, mechanistic properties and biotechnological potential

Chitin derivatives, chitosan and substituted chito-oligosaccharides have a wide spectrum of applications ranging from medicine to cosmetics and dietary supplements. With advancing knowledge about the substrate-binding properties of chitinases, enzyme-based production of these biotechnologically relevant sugars from biological resources is becoming increasingly interesting. Fungi have high numbers of glycoside hydrolase family 18 chitinases with different substrate-binding site architectures. As presented in this review, the large diversity of fungal chitinases is an interesting starting point for protein engineering. In this review, recent data about the architecture of the substrate-binding clefts of fungal chitinases, in connection with their hydrolytic and transglycolytic abilities, and the development of chitinase inhibitors are summarized. Furthermore, the biological functions of chitinases, chitin and chitosan utilization by fungi, and the effects of these aspects on biotechnological applications, including protein overexpression and autolysis during industrial processes, are discussed in this review

Synthesis and evaluation of a 99mTechnetium labeled chitin-binding protein as potential specific radioligand for the detection of fungal infections in mice.

AIM: Radiopharmaceuticals can be used to exploit differences between microorganisms in order to distinguish fungal from bacterial infection. Chitin, abundant in the cell wall of fungi, is not present in mammalian or bacterial cells and therefore represents a highly specific target to localize fungal infection. In this study, we have examined the potential of chitin-binding protein (CBP21) from Serratia marcescens as a specific radiotracer for the detection of invasive fungal infections. METHODS: CBP21 was labeled with 99mTc via hydrazinonicotinamide (HYNIC) and its characteristics were analyzed. In vitro binding studies with polymorphic chitin forms and microorganisms (fungi as well as bacteria) were performed. In vivo biodistribution of the compound was studied in immunocompromised mice with bacterial and fungal infections in the left and right thigh muscle, respectively, using 99mTc-HYNIC-myoglobin as size-matched control and 67Ga-citrate as positive control. Scintigraphic images were acquired at 1 and 7 h postinjection of the tracer. RESULTS: 99mTc-HYNIC-CBP21 was labeled with a radiochemical yield of 61% and a specific activity of 22.3 MBq/nmol. Highest in vitro binding percentages were found with beta-chitin (86.8+/-2.4%). Binding interactions to fungi were higher than to bacteria (P<0.05). In vivo, best ratios of fungal infection versus bacterial infection were seen at 5 and 7 h (3.6+/-1.2 and 2.9+/-1.4, respectively) postinjection of the tracer. Maximum uptake of the tracer in fungal infections (0.63+/-0.11%ID/g) at 7 h was significantly (P<0.05) higher than uptake seen in bacterial infections (0.34+/-0.11%ID/g) or the uptake of 99mTc-HYNIC-myoglobin (P<0.05) in the same infections (0.35+/-0.11%ID/g, respectively, 0.3+/-0.01%ID/g). CONCLUSIONS: This study shows that 99mTc-HYNIC-CBP21 is able to specifically interact with chitin in vitro. Scintigraphy and postmortem in vivo data indicate that 99mTc-HYNIC-CBP21 is able to distinguish fungal infection from bacterial infection probably due to a specific interaction of the protein with the chitin in the fungal cell wall