Crystallization and preliminary crystallographic analysis of LipC12, a true lipase isolated through a metagenomics approach.

LipC12, a true lipase from family I.1 of bacterial lipases which was previously isolated through a metagenomics approach, contains 293 amino acids. Among lipases of known three-dimensional structure, it has a sequence identity of 47% to the lipase from Pseudomonas aeruginosa PAO1. Recombinant N-terminally His(6)-tagged LipC12 protein was expressed in Escherichia coli, purified in a homogenous form and crystallized in several conditions, with the best crystals being obtained using 2.0 M sodium formate and 0.1 M bis-tris propane pH 7.0. X-ray diffraction data were collected to 2.70 Å resolution. The crystals belonged to the tetragonal space group P4(1)22, with unit-cell parameters a = b = 58.62, c = 192.60 Å.

Preliminary X-ray crystallographic studies of a tetrameric phospholipase A2 formed by two isoforms of crotoxin B from Crotalus durissus terrificus venom.

Crotoxin B is a basic phospholipase A2 found in the venom of Crotalus durissus terrificus and is one of the subunits that constitute crotoxin. This heterodimeric toxin, which is the main component of C. d. terrificus venom, is completed by an acidic, nontoxic and non-enzymatic component (crotoxin A) and is involved in important envenomation effects, such as neurological disorders, myotoxicity and renal failure. Although crotoxin was first crystallized in 1938, no crystal structure is currently available for crotoxin, crotoxin A or crotoxin B. In this work, the crystallization, X-ray diffraction data collection to 2.28 A resolution and molecular-replacement solution of a novel tetrameric complex formed by two dimers of crotoxin B isoforms (CB1 and CB2) is presented.

Crystal structure of adenine phosphoribosyltransferase from Leishmania tarentolae: potential implications for APRT catalytic mechanism.

The three-dimensional structure of Leishmania tarentolae adenine phosphoribosyltransferase (APRT) in complex with adenosine-5-monophosphate (AMP) and a phosphate ion has been solved. Refinement against X-ray diffraction data extending to 2.2-A resolution led to a final crystallographic R factor of 18.3%. Structural comparisons amongst this APRT enzyme and other 'type I' PRTases whose structures have been determined reveal several important features of the PRTases catalytic mechanism. Based on structural superpositions and molecular interaction potential calculations, it was possible to suggest that the PRPP is the first substrate to bind, while the AMP is the last product to leave the active site, in accordance to recent kinetic studies performed with the Leishmania donovani APRT.

Purification, biochemical characterisation and partial primary structure of a new alpha-amylase inhibitor from Secale cereale (rye).

Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by the observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Better understanding of this specificity depends on modelling studies based on ample structural and biochemical information. A new member of the alpha-amylase inhibitor family of cereal endosperm has been purified from rye using two ionic exchange chromatography steps. It has been characterised by mass spectrometry, inhibition assays and N-terminal protein sequencing. The results show that the inhibitor has a monomer molecular mass of 13,756 Da, is capable of dimerisation and is probably glycosylated. The inhibitor has high homology with the bifunctional alpha-amylase/trypsin inhibitors from barley and wheat, but much poorer homology with other known inhibitors from rye. Despite the homology with bifunctional inhibitors, this inhibitor does not show activity against mammalian or insect trypsin, although activity against porcine pancreatic, human salivary, Acanthoscelides obtectus and Zabrotes subfasciatus alpha-amylases was observed. The inhibitor is more effective against insect alpha-amylases than against mammalian enzymes. It is concluded that rye contains a homologue of the bifunctional alpha-amylase/trypsin inhibitor family without activity against trypsins. The necessity of exercising caution in assigning function based on sequence comparison is emphasised.

Crystallization and preliminary crystallographic studies of alpha-amylase inhibitor from wheat (Triticum aestivum).

Crystals of the human salivary alpha-amylase inhibitor from wheat have been obtained. A native data set was collected to 2.1 A resolution with 90% completeness at laboratory sources. The crystals belong to the trigonal system, space group P3(1) (or enantiomer) with a = b = 79.31, c = 60.56 A. Crystal density analysis and self-rotation function studies suggest the presence of four subunits in the asymmetric unit.