High-level expression of the native barley alpha-amylase/subtilisin inhibitor in Pichia pastoris.

An expression system for high-level expression of the native Hordeum vulgare alpha-amylase/subtilisin inhibitor (BASI) has been developed in Pichia pastoris, using the methanol inducible alcohol oxidase 1 (AOX1) promoter. To optimize expression, two codon-optimized coding regions have been designed and expressed alongside the wild-type coding region. To ensure secretion of the native mature protein, a truncated version of the alpha mating factor secretion signal from Saccharomyces cerevisiae was used. In order to be able to compare expression levels from different clones, single insertion transformants generated by gene replacement of the AOX1 gene was selected by PCR screening. Following methanol induction, expression levels reached 125 mgL(-1) from the wild-type coding region while expression from the two codon-optimized variants reached 65 and 125 mgL(-1), respectively. The protein was purified and characterized by Edman degradation, liquid chromatography mass spectrometry and insoluble blue starch assay, and was shown to possess the same characteristics as wild-type protein purified from barley grains.

Crystallization and preliminary X-ray diffraction analysis of the small laccase from Streptomyces coelicolor.

The small bacterial laccase from the actinobacterium Streptomyces coelicolor which lacks the second of the three domains of the laccases structurally characterized to date was crystallized. This multi-copper phenol oxidase crystallizes in a primitive tetragonal lattice, with unit-cell parameters a = b = 179.8, c = 175.3 A. The crystals belong to either space group P4(1)2(1)2 or P4(3)2(1)2. The self-rotation function shows the presence of a noncrystallographic threefold axis in the structure. Phases will be determined from the anomalous signal of the natively present copper ions.

Structure of two fungal beta-1,4-galactanases: searching for the basis for temperature and pH optimum.

beta-1,4-Galactanases hydrolyze the galactan side chains that are part of the complex carbohydrate structure of the pectin. They are assigned to family 53 of the glycoside hydrolases and display significant variations in their pH and temperature optimum and stability. Two fungal beta-1,4-galactanases from Myceliophthora thermophila and Humicola insolens have been cloned and heterologously expressed, and the crystal structures of the gene products were determined. The structures are compared to the previously only known family 53 structure of the galactanase from Aspergillus aculeatus (AAGAL) showing approximately 56% identity. The M. thermophila and H. insolens galactanases are thermophilic enzymes and are most active at neutral to basic pH, whereas AAGAL is mesophilic and most active at acidic pH. The structure of the M. thermophila galactanase (MTGAL) was determined from crystals obtained with HEPES and TRIS buffers to 1.88 A and 2.14 A resolution, respectively. The structure of the H. insolens galactanase (HIGAL) was determined to 2.55 A resolution. The thermostability of MTGAL and HIGAL correlates with increase in the protein rigidity and electrostatic interactions, stabilization of the alpha-helices, and a tighter packing. An inspection of the active sites in the three enzymes identifies several amino acid substitutions that could explain the variation in pH optimum. Examination of the activity as a function of pH for the D182N mutant of AAGAL and the A90S/ H91D mutant of MTGAL showed that the difference in pH optimum between AAGAL and MTGAL is at least partially associated with differences in the nature of residues at positions 182, 90, and/or 91.

Activity of three ß-1,4-galactanases on small chromogenic substrates.

ß-1,4-Galactanases belong to glycoside hydrolase family GH 53 and degrade galactan and arabinogalactan side chains of the complex pectin network in plant cell walls. Two fungal ß-1,4-galactanases from Aspergillus aculeatus, Meripileus giganteus and one bacterial enzyme from Bacillus licheniformis have been kinetically characterized using the chromogenic substrate analog 4-nitrophenyl ß-1,4-d-thiogalactobioside synthesized by the thioglycoligase approach. Values of k(cat)/K(m) for this substrate with A. aculeatus ß-1,4-galactanase at pH 4.4 and for M. giganteus ß-1,4-galactanase at pH 5.5 are 333M(-1)s(-1) and 62M(-1)s(-1), respectively. By contrast the B. licheniformis ß-1,4-galactanase did not hydrolyze 4-nitrophenyl ß-1,4-d-thiogalactobioside. The different kinetic behavior observed between the two fungal and the bacterial ß-1,4-galactanases can be ascribed to an especially long loop 8 observed only in the structure of B. licheniformis ß-1,4-galactanase. This loop contains substrate binding subsites -3 and -4, which presumably cause B. licheniformis ß-1,4-galactanase to bind 4-nitrophenyl -1,4-ß-d-thiogalactobioside non-productively. In addition to their cleavage of 4-nitrophenyl -1,4-ß-d-thiogalactobioside, the two fungal enzymes also cleaved the commercially available 2-nitrophenyl-1,4-ß-d-galactopyranoside, but kinetic parameters could not be determined because of transglycosylation at substrate concentrations above 4mM.

The structure of the small laccase from Streptomyces coelicolor reveals a link between laccases and nitrite reductases.

The X-ray structure of the two-domain laccase (small laccase) from Streptomyces coelicolor A3(2) was solved at 2.7-A resolution. The enzyme differs significantly from all laccases studied structurally so far. It consists of two domains and forms trimers and hence resembles the quaternary structure of nitrite reductases or ceruloplasmins more than that of large laccases. There are three trinuclear copper clusters in the enzyme localized between domains 1 and 2 of each pair of neighbor chains. In this way, a similar geometry of the active site as seen in large laccases is ensured, albeit by different arrangements of domains and protein chains. Three copper ions of type 1 lie close to one another near the surface of the central part of the trimer, and, effectively, a trimeric substrate binding site is formed in their vicinity.

Structural and mutational analyses of the interaction between the barley alpha-amylase/subtilisin inhibitor and the subtilisin savinase reveal a novel mode of inhibition.

Subtilisins represent a large class of microbial serine proteases. To date, there are three-dimensional structures of proteinaceous inhibitors from three families in complex with subtilisins in the Protein Data Bank. All interact with subtilisin via an exposed loop covering six interacting residues. Here we present the crystal structure of the complex between the Bacillus lentus subtilisin Savinase and the barley alpha-amylase/subtilisin inhibitor (BASI). This is the first reported structure of a cereal Kunitz-P family inhibitor in complex with a subtilisin. Structural analysis revealed that BASI inhibits Savinase in a novel way, as the interacting loop is shorter than loops previously reported. Mutational analysis showed that Thr88 is crucial for the inhibition, as it stabilises the interacting loop through intramolecular interactions with the BASI backbone.