High-resolution native and complex structures of thermostable beta-mannanase from Thermomonospora fusca - substrate specificity in glycosyl hydrolase family 5.

BACKGROUND: . beta-Mannanases hydrolyse the O-glycosidic bonds in mannan, a hemicellulose constituent of plants. These enzymes have potential use in pulp and paper production and are of significant biotechnological interest. Thermostable beta-mannanases would be particularly useful due to their high temperature optimum and broad pH tolerance. The thermophilic actinomycete Thermomonospora fusca secretes at least one beta-mannanase (molecular mass 38 kDa) with a temperature optimum of 80 degreesC. No three-dimensional structure of a mannan-degrading enzyme has been reported until now. RESULTS: . The crystal structure of the thermostable beta-mannanase from T. fusca has been determined by the multiple isomorphous replacement method and refined to 1.5 A resolution. In addition to the native enzyme, the structures of the mannotriose- and mannohexaose-bound forms of the enzyme have been determined to resolutions of 1.9 A and 1.6 A, respectively. CONCLUSIONS: . Analysis of the -1 subsite of T. fusca mannanase reveals neither a favourable interaction towards the axial HO-C(2) nor a discrimination against the equatorial hydroxyl group of gluco-configurated substrates. We propose that selectivity arises from two possible mechanisms: a hydrophobic interaction of the substrate with Val263, conserved in family 5 bacterial mannanases, which discriminates between the different conformations of the hydroxymethyl group in native mannan and cellulose; and/or a specific interaction between Asp259 and the axial hydroxyl group at the C(2) of the substrate in the -2 subsite. Compared with the catalytic clefts of family 5 cellulases, the groove of T. fusca mannanase has a strongly reduced number of aromatic residues providing platforms for stacking with the substrate. This deletion of every second platform is in good agreement with the orientation of the axial hydroxyl groups in mannan.

Crystallization and preliminary crystallographic analysis of two beta-mannanase isoforms from Thermomonospora fusca KW3.

Three beta-mannanase isoforms were isolated from the supernatant of a thermophilic actinomycete culture from Thermomonospora fusca KW3. Two of the isoforms (Q1, Q 1.1) were crystallized by the hanging-drop method at room temperature using ammonium sulfate as a precipitant. The isoforms form rod-shaped colorless crystals. Both belong to the orthorhombic space group P2(1)2(1)2(1). The cell dimensions are a = 46.7, b = 61.1, and c = 128.2 A for isoform Q1, and a = 43.8, b = 46.2, and c = 132.8 A for isoform Q1.1. The asymmetric unit of either isoform contains one mannanase molecule. Native data have been collected to 2.2 A resolution for Q1 and to 1.65 A resolution for Q1.1 using synchrotron radiation.

Structure elucidation of beta-mannanase: from the electron-density map to the DNA sequence.

The crystal structure of affinity-purified Thermomonospora fusca beta-mannanase has been solved despite the lack of the major part of the amino-acid sequence. A high-quality electron-density map allowed the identification of a stretch of eight amino acids close to the C-terminus which was used to design a degenerate downstream PCR primer. Together with a specific primer previously derived from the N-terminus, 95.7% of the mannanase gene sequence was obtained from genomic T. fusca DNA by PCR. The structure-derived sequence was then compared with the DNA-derived sequence and corrected when necessary. Applying the presented protocol, there was no need to manually build a model at an early stage of structure determination, an erroneous and tedious process, especially in the absence of the amino-acid sequence. Using the DNA sequence information and the current version of ARP/wARP, 281 residues, or 93% of the polypeptide chain (including side chains), were built and refined to an R factor of 16.5% without any manual intervention.

Matrix methods for solving protein substructures of chlorine and sulfur from anomalous data.

The weak signal obtained from the anomalous scattering (at lambda = 1.54 A) of naturally occurring elements such as sulfur, phosphorus and ordered solvent chloride ions is used to determine the atomic positions of these atoms. Two examples are discussed: the sulfur and chlorine substructure of tetragonal hen egg-white lysozyme and an oligonucleotide containing ten P atoms. The substructure of lysozyme was also solved from Cu K(alpha) radiation data collected on a standard rotating-anode generator. The results presented here are an illustration of the power of the matrix methods, which are to be implemented in next distribution of the direct methods package CRUNCH.