Susan Mampusti Madrid

Characterization of a new antifungal chitin-binding peptide from sugar beet leaves

The intercellular washing fluid (IWF) from leaves of sugar beet (Beta vulgaris L.) contains a number of proteins exhibiting in vitro antifungal activity against the devastating leaf pathogen Cercospora beticola (Sacc.). Among these, a potent antifungal peptide, designated IWF4, was identified. The 30-amino-acid residue sequence of IWF4 is rich in cysteines (6) and glycines (7) and has a highly basic isoelectric point. IWF4 shows homology to the chitin-binding (hevein) domain of chitin-binding proteins, e.g. class I and IV chitinases. Accordingly, IWF4 has a strong affinity to chitin. Notably, it binds chitin more strongly than the chitin-binding chitinases. A full-length IWF4 cDNA clone was obtained that codes for a preproprotein of 76 amino acids containing an N-terminal putative signal peptide of 21 residues, followed by the mature IWF4 peptide of 30 residues, and an acidic C-terminal extension of 25 residues. IWF4 mRNA is expressed in the aerial parts of the plant only, with a constitutive expression in young and mature leaves and in young flowers. No induced expression of IWF4 protein or mRNA was detected during infection with C. beticola or after treatment with 2,6-dichloroisonicotinic acid, a well-known inducer of resistance in plants.

New antifungal proteins from sugar beet (Beta vulgaris L.) showing homology to non-specific lipid transfer proteins

Two novel, nearly identical antifungal proteins, IWF1 and IWF2, were isolated from the intercellular washing fluid (IWF) of sugar beet leaves. The proteins were purified to homogeneity and their amino acid sequences were determined. They are basic, monomeric proteins of 91 amino acid residues, 89 of which are identical. Both proteins show strongin vitro antifungal activity againstCercospora beticola, the casual agent of leaf spot disease in sugar beet. Based on primary sequence homology, including the presence of 8 conserved cysteine residues, IWF1 and IWF2 are related to the family of plant non-specific lipid transfer proteins (nsLTPs). Antibodies were raised against IWF2 after conjugation to diphtheria toxoid. The amino acid sequence data was used to generate a polymerase chain reaction (PCR) clone, employed for the isolation of a cDNA clone encoding a closely related isoform IWFA, which differs from IWF1 by two amino acid substitutions only. The induction and subcellular localization of these proteins were studied by western and northern blotting analyses after treatment with 2,6-dichloroisonicotinic acid (INA), a compound capable of inducing resistance againstC. beticola, and after fungal infection. The following observations were made: (1) the proteins were present in leaves of non-INA-treated and uninfected control plants, (2) they were only slightly induced by INA treatment and during infection withC. beticola, and (3) they were present both intra- and extracellularly. However, their strong antifungal potentials together with immunohistological investigations, the proteins accumulating in contact with the fungus and in autolysing cells, suggested a role of these proteins in plant defence. Finally, immunohistology revealed a remarkable expression pattern of the IWF1 and IWF2 proteins, or serologically related proteins, in sugar beet styles, in that single or a few scattered papillae and a few cells in the lower transmitting tissue strongly and specifically reacted with the antibody.

HACA, the transcriptional activator of the unfolded protein response (UPR) in Aspergillus niger, binds to partly palindromic UPR elements of the consensus sequence 5'-CAN(G/A)NTGT/GCCT-3'.

The promoters of UPR target genes contain an unfolded protein response element (UPRE), which confers the stress inducibility to the gene, via an interaction with the transcription activator HACA. In the promoters of the Aspergillus ER-stress responsive genes bipA, cypB, pdiA, prpA, tigA, and hacA, a consensus sequence was identified, which was located close to the transcription start site of the gene (<81 bp), and corresponds to the sequence CAN(G/A)NTGT/GCCT. The UPRE is a partly palindromic sequence around a dispensable spacer nucleotide, followed by four highly conserved bases. By an in vitro selection procedure, an optimal binding site for HACA was isolated. This sequence, ACACGTGTCCT, resembles the UPRE but lacks the spacer nucleotide. It has a much higher binding affinity than the identified UPREs, and in vivo it behaves as a more powerful cis-acting element.

Crystal Structure of α-1,4-Glucan Lyase, a Unique Glycoside Hydrolase Family Member with a Novel Catalytic Mechanism

Background: α-1,4-Glucan lyase (GLase) is a glycoside hydrolase family member that degrades starch via an elimination reaction. Results: Crystal structures of GLase with covalently bound inhibitors show that the catalytic nucleophile can abstract the proton. Conclusion: The nucleophile has a dual function, acting successively as nucleophile and base. Significance: A single substitution converts a glycoside hydrolase into a lyase. α-1,4-Glucan lyase (EC 4.2.2.13) from the red seaweed Gracilariopsis lemaneiformis cleaves α-1,4-glucosidic linkages in glycogen, starch, and malto-oligosaccharides, yielding the keto-monosaccharide 1,5-anhydro-d-fructose. The enzyme belongs to glycoside hydrolase family 31 (GH31) but degrades starch via an elimination reaction instead of hydrolysis. The crystal structure shows that the enzyme, like GH31 hydrolases, contains a (β/α)8-barrel catalytic domain with B and B′ subdomains, an N-terminal domain N, and the C-terminal domains C and D. The N-terminal domain N of the lyase was found to bind a trisaccharide. Complexes of the enzyme with acarbose and 1-dexoynojirimycin and two different covalent glycosyl-enzyme intermediates obtained with fluorinated sugar analogues show that, like GH31 hydrolases, the aspartic acid residues Asp553 and Asp665 are the catalytic nucleophile and acid, respectively. However, as a unique feature, the catalytic nucleophile is in a position to act also as a base that abstracts a proton from the C2 carbon atom of the covalently bound subsite −1 glucosyl residue, thus explaining the unique lyase activity of the enzyme. One Glu to Val mutation in the active site of the homologous α-glucosidase from Sulfolobus solfataricus resulted in a shift from hydrolytic to lyase activity, demonstrating that a subtle amino acid difference can promote lyase activity in a GH31 hydrolase.

Crystal Structure of Bifunctional Aldos-2-Ulose Dehydratase/Isomerase from Phanerochaete Chrysosporium with the Reaction Intermediate Ascopyrone M.

The enzyme aldos-2-ulose dehydratase/isomerase (AUDH) participates in carbohydrate secondary metabolism, catalyzing the conversion of glucosone and 1,5-d-anhydrofructose to the secondary metabolites cortalcerone and microthecin, respectively. AUDH is a homo-dimeric enzyme with subunits of 900 amino acids. The subunit consists of a seven-bladed β-propeller domain, two cupin folds and a C-terminal lectin domain. AUDH contains a structural Zn(2+) and Mg(2+) located in loop regions and two zinc ions at the bottom of two putative active-site clefts in the propeller and the cupin domain, respectively. Catalysis is dependent on these two zinc ions, as their specific removal led to loss of enzymatic activity. The structure of the Zn(2)(+)-depleted enzyme is very similar to that of native AUDH, and structural changes upon metal removal as the cause for the catalytic deficiencies can be excluded. The complex with the reaction intermediate ascopyrone M shows binding of this compound at two different sites, with direct coordination to Zn(2+) in the propeller domain and as second sphere ligand of the metal ion in the cupin domain. These observations suggest that the two reactions of AUDH might be catalyzed in two different active sites, about 60 Å apart. The dehydration reaction most likely follows an elimination mechanism, where Zn(2+) acts as a Lewis acid polarizing the C2 keto group of 1,5-d-anhydrofructose. Abstraction of the proton at the C3 carbon atom and protonation of the leaving group, the C4 hydroxyl moiety, could potentially be catalyzed by the side chain of the suitably positioned residue His155.