Crystal structure of fungal tannase from Aspergillus niger

Tannases are serine esterases that were first discovered in fungi more than one and half centuries ago. They catalyze the hydrolysis of the gallolyl ester bonds in gallotannins to release gallic acid, which is an important intermediate in the chemical and pharmaceutical industries. Since their discovery, fungal tannases have found wide industrial applications, although there is scarce knowledge about these enzymes at the molecular level, including their catalytic and substrate-binding sites. While this lack of knowledge hinders engineering efforts to modify the enzymes, many tannases have been isolated from various fungal strains in a search for the desired enzymatic properties. Here, the first crystal structure of a fungal tannase, that from Aspergillus niger, is reported. The enzyme possesses a typical α/β-hydrolase-fold domain with a large inserted cap domain, which together form a bowl-shaped hemispherical shape with a surface concavity surrounded by N-linked glycans. Gallic acid is bound at the junction of the two domains within the concavity by forming two hydrogen-bonding networks with neighbouring residues. One is formed around the carboxyl group of the gallic acid and involves residues from the hydrolase-fold domain, including those from the catalytic triad, which consists of Ser206, His485 and Asp439. The other is formed around the three hydroxyl groups of the compound, with the involvement of residues mainly from the cap domain, including Gln238, Gln239, His242 and Ser441. Gallic acid is bound in a sandwich-like mode by forming a hydrophobic contact with Ile442. All of these residues are found to be highly conserved among fungal and yeast tannases.

Expression, purification, crystallization and preliminary X-ray analysis of tannase fromLactobacillus plantarum

Tannase catalyses the hydrolysis of the galloyl ester bond of tannins to release gallic acid. It belongs to the serine esterases and has wide applications in the food, feed, beverage, pharmaceutical and chemical industries. The tannase fromLactobacillus plantarumwas cloned, expressed and purified. The protein was crystallized by the sitting-drop vapour-diffusion method with microseeding. The crystals belonged to space groupP1, with unit-cell paramtersa= 46.5,b= 62.8,c= 83.8 Å, α = 70.4, β = 86.0, γ = 79.4°. Although the enzyme exists mainly as a monomer in solution, it forms a dimer in the asymmetric unit of the crystal. The crystals diffracted to beyond 1.60 Å resolution using synchrotron radiation and a complete data set was collected to 1.65 Å resolution.

An important role for secreted esterase in disease establishment of the wheat powdery mildew fungus Blumeria graminis f. sp.tritici

The activity of esterase secreted by conidia of wheat powdery mildew fungus, Blumeria graminis f. sp. tritici, was assayed using indoxyl acetate hydrolysis, which generates indigo blue crystals. Mature, ungerminated, and germinating conidia secrete esterase(s) on artificial media and on plant leaf surfaces. The activity of these esterases was inhibited by diisopropyl fluorophosphate, which is selective for serine esterases. When conidia were inoculated on wheat leaves pretreated with diisopropyl fluorophosphate, both appressorial germ tube differentiation and symptom development were significantly impaired, indicating an important role of secreted serine esterases in wheat powdery mildew disease establishment.

Lipases and Related Molecules in Cancer

Lipases are enzymes that catalyze the hydrolysis of lipids. Based on protein structures and sequences, lipases can be classified into different protein families. The majority of conventional mammalian lipases are members of the protein super-families of serine esterases and alpha-beta hydrolases. Differential expression of lipases and related alpha-beta hydrolases in tumor cells has been observed. The physiological or patho-physiological functions of these tumor related enzymes are largely unknown. However, lipases are not only involved in energy metabolism but also in the metabolism of bioactive molecules, e.g. phosphatidic acid or arachidonic acid, suggesting that tumor-specifically expressed lipases might be interesting targets for the development of future treatment strategies. Moreover, independent of the patho-physiological function, tumor associated lipases can serve as targets for immunological treatment strategies. In addition, lipases with exclusive expression in single tumor entities can serve as potential diagnostic targets.

Purification and Characterization of Mycobacterial Phospholipase A: an Activity Associated with Mycobacterial Cutinase

ABSTRACT We describe mycobacterial phospholipase A activity (MPLA) and, using reverse genetics, have associated this activity with putative mycobacterial cutinase. PLAs, which hydrolyze fatty acids on phospholipids, play a significant role in human inflammatory states and disease pathogenesis. In prokaryotes, the recognition of their role in virulence is more recent. Cutinases are serine esterases whose primary substrate is cutin, the waxy exterior layer of plants. Mycobacterium tuberculosis has maintained seven putative cutinases, though it should not encounter cutin; we demonstrate that known cutinases and MPLA cleave phospholipids in a PLA-type manner and also hydrolyze Tween. We analyzed cutinase motifs in mycobacteria and found the motif very prevalent. All mycobacteria tested had MPLA activity. These studies suggest an alternative use for putative cutinases by the M. tuberculosis group that is likely related to MPLA activity and lipid metabolism.