Structural and electronic determinants of lytic polysaccharide monooxygenase reactivity on polysaccharide substrates.

Lytic polysaccharide monooxygenases (LPMOs) are industrially important copper-dependent enzymes that oxidatively cleave polysaccharides. Here we present a functional and structural characterization of two closely related AA9-family LPMOs from Lentinus similis (LsAA9A) and Collariella virescens (CvAA9A). LsAA9A and CvAA9A cleave a range of polysaccharides, including cellulose, xyloglucan, mixed-linkage glucan and glucomannan. LsAA9A additionally cleaves isolated xylan substrates. The structures of CvAA9A and of LsAA9A bound to cellulosic and non-cellulosic oligosaccharides provide insight into the molecular determinants of their specificity. Spectroscopic measurements reveal differences in copper co-ordination upon the binding of xylan and glucans. LsAA9A activity is less sensitive to the reducing agent potential when cleaving xylan, suggesting that distinct catalytic mechanisms exist for xylan and glucan cleavage. Overall, these data show that AA9 LPMOs can display different apparent substrate specificities dependent upon both productive protein-carbohydrate interactions across a binding surface and also electronic considerations at the copper active site.

Gene cloning, sequencing, and characterization of a family 9 endoglucanase (CelA) with an unusual pattern of activity from the thermoacidophile Alicyclobacillus acidocaldarius ATCC27009.

A gene encoding a beta-1,4-glucanase (CelA) belonging to subfamily E1 of family 9 of glycoside hydrolases was cloned and sequenced from the gram-positive thermoacidophile Alicyclobacillus acidocaldarius strain ATCC27009. The translated protein contains an immunoglobulin-like domain but lacks a cellulose-binding domain. The enzyme, when overproduced in Escherichia coli and purified, displayed a temperature optimum of 70 degrees C and a pH optimum of 5.5. CelA contained one zinc and two calcium atoms. Calcium and zinc are likely to be important for temperature stability. The enzyme was most active against substrates containing beta-1,4-linked glucans (lichenan and carboxy methyl cellulose), but also exhibited activity against oat spelt xylan. A striking pattern of hydrolysis on p-nitrophenyl-glycosides was observed, with highest activity on the cellobioside derivative, some on the cellotetraoside derivative, and none on the glucoside and cellotrioside derivatives. Unmodified cellooligosaccharides were also hydrolyzed by CelA. No signal peptide for transport across the cytoplasmic membrane was detected. This, together with the substrate specificity displayed, near neutral pH optimum and irreversible inactivation at low pH, suggests a role for CelA as a cytoplasmic enzyme for the degradation of imported oligosaccharides.

Substrate specificity and subsite mobility in T. aurantiacus xylanase 10A.

The substrate specificity of Thermoascus aurantiacus xylanase 10A (TAX) has been investigated both biochemically and structurally. High resolution crystallographic analyses at 291 K and 100 K of TAX complexes with xylobiose show that the ligand is in its alpha anomeric conformation and provide a rationale for specificity on p-nitrophenyl glycosides at the -1 and -2 subsites. Trp 275, which is disordered in uncomplexed structures, is stabilised by its interaction with xylobiose. Two structural subsets in family 10 are identified, which differ by the presence or absence of a short helical stretch in the eighth betaalpha-loop of the TIM barrel, the loop bearing Trp 275. This structural difference is discussed in the context of Trp 275 mobility and xylanase function.

Crystallization and preliminary X-ray analysis of maltose O-acetyltransferase.

Maltose O-acetyltransferase (Mac) is a member of the hexapeptide-repeat family of enzymes, which contains proteins with left-handed parallel beta-helix architecture forming homotrimers. Diffraction data for four well diffracting crystal forms were collected. Crystal form I diffracted beyond 1.53 A resolution but was perfectly merohedrally twinned with an apparent space group P622. Crystal forms II and III (space groups R3 and C2, respectively) could be obtained under very similar conditions by adjusting the buffer pH differently. Crystal forms II and III had several monomers in the asymmetric unit and were difficult to derivatize. However, during soaking with trimethyl lead acetate, the form III crystals dissolved and crystals with a different habit and space group grew in their place (form IV). In three of the crystal forms, a ladder of peaks was visible in the native Patterson maps along the c axis. These peaks were interpreted as corresponding to the vectors between the beta-strands in the turns of the beta-helix. Crystal form IV is suitable for structure determination of Mac exploiting the anomalous scattering of lead.

High resolution structure and sequence of T. aurantiacus xylanase I: implications for the evolution of thermostability in family 10 xylanases and enzymes with (beta)alpha-barrel architecture.

Xylanase I is a thermostable xylanase from the fungus Thermoascus aurantiacus, which belongs to family 10 in the current classification of glycosyl hydrolases. We have determined the three-dimensional X-ray structure of this enzyme to near atomic resolution (1.14 A) by molecular replacement, and thereby corrected the chemically determined sequence previously published. Among the five members of family 10 enzymes for which the structure has been determined, Xylanase I from T. aurantiacus and Xylanase Z from C. thermocellum are from thermophilic organisms. A comparison with the three other available structures of the family 10 xylanases from mesophilic organisms suggests that thermostability is effected mainly by improvement of the hydrophobic packing, favorable interactions of charged side chains with the helix dipoles and introduction of prolines at the N-terminus of helices. In contrast to other classes of proteins, there is very little evidence for a contribution of salt bridges to thermostability in the family 10 xylanases from thermophiles. Further analysis of the structures of other proteins from thermophiles with eight-fold (beta)alpha-barrel architecture suggests that favorable interactions of charged side chains with the helix dipoles may be a common way in which thermophilic proteins with this fold are stabilized. As this is the most common type of protein architecture, this finding may provide a useful guide for site-directed mutagenesis aimed to improve the thermostability of (beta)alpha-barrel proteins. Proteins 1999;36:295-306.

Crystallization and preliminary X-ray analysis of the major endoglucanase from Thermoascus aurantiacus.

The major endoglucanase (35 kDa) from the thermophilic fungus Thermoascus aurantiacus has been purified from culture filtrates using an affinity method and the sequence for 35 N-terminal amino acids determined. This has allowed assignment of the enzyme to subtype A6 of family 5 endoglucanases. The enzyme has been crystallized as thick plates by the hanging-drop method using ammonium sulfate as precipitant. The crystals belong to space group P2(1)2(1)2(1) with cell edges a = 76.4, b = 85.7 and c = 89.5 A, with two molecules in the asymmetric unit, and diffract to 1.62 A resolution using synchrotron radiation. The structure will be solved by isomorphous replacement.

Inhibition of sperm-zona binding by suramin, a potential 'lead' compound for design of new anti-fertility agents.

Progress in developing new contraceptive agents, particularly for the male, is extremely slow. Here, we report on a novel property of the anti-trypanosomal drug suramin: the ability to act at the sperm-egg interface and prevent fertilization. Suramin is a polysulphonated compound that inhibits binding of capacitated mouse spermatozoa to the zona pellucida in vitro with an IC50 of 12.4 microM. The drug is only effective at the time of fertilization and is not inhibitory if gametes are pre-treated separately. Autoradiographic localization of suramin binding sites on mouse, boar and human spermatozoa shows that they are intracellular, principally in the head region. The sperm protein recognized by suramin has been identified as proacrosin which is known to interact with sulphate groups on zona glycoproteins. Zona pellucida glycoproteins do not bind suramin, suggesting that the drug blocks the ability of proacrosin/acrosin, exposed during the acrosome reaction, to mediate the secondary binding phase of spermatozoa to the zona during fertilization. Structure-based design studies have the potential to generate safe suramin mimetics that would form the basis for a new generation of non-steroidal contraceptive agents.

Beta-glucosidase, beta-galactosidase, family A cellulases, family F xylanases and two barley glycanases form a superfamily of enzymes with 8-fold beta/alpha architecture and with two conserved glutamates near the carboxy-terminal ends of beta-strands four and seven.

Comparison of the recently determined crystal structures Pseudomonas fluorescens subsp. cellulosa family F xylanase, (1-3)-beta-glucanase and (1-3,1-4)-beta-glucanase and the catalytic domain of E. coli beta-galactosidase reveals that they belong to a superfamily of 8-fold beta/alpha-barrels with similar amino acid residues at their active sites. In the three families that these enzymes represent, the nucleophile is a glutamate, which is located close to the carboxy-terminus of beta-strand seven. In addition all three enzymes have the sequence asparagine-glutamate close to the carboxy-terminus of beta-strand four. This glutamate has been identified as the acid/base in the family F xylanases and is essential for catalysis in beta-galactosidase. We suggest that the equivalent residue in the barley glucanases is the acid/base. Analysis of the sequences of family 1 beta-glucosidases and family 5 cellulases shows that these enzymes also belong to this superfamily which we call the 4/7 superfamily.

Structure of the catalytic core of the family F xylanase from Pseudomonas fluorescens and identification of the xylopentaose-binding sites.

BACKGROUND: Sequence alignment suggests that xylanases evolved from two ancestral proteins and therefore can be grouped into two families, designated F and G. Family F enzymes show no sequence similarity with any known structure and their architecture is unknown. Studies of an inactive enzyme-substrate complex will help to elucidate the structural basis of binding and catalysis in the family F xylanases. RESULTS: We have therefore determined the crystal structure of the catalytic domain of a family F enzyme, Pseudomonas fluorescens subsp. cellulosa xylanase A, at 2.5 A resolution and a crystallographic R-factor of 0.20. The structure was solved using an engineered catalytic core in which the nucleophilic glutamate was replaced by a cysteine. As expected, this yielded both high-quality mercurial derivatives and an inactive enzyme which enabled the preparation of the inactive enzyme-substrate complex in the crystal. We show that family F xylanases are eight-fold alpha/beta-barrels (TIM barrels) with two active-site glutamates, one of which is the nucleophile and the other the acid-base. Xylopentaose binds to five subsites A-E with the cleaved bond between subsites D and E. Ca2+ binding, remote from the active-site glutamates, stabilizes the structure and may be involved in the binding of extended substrates. CONCLUSIONS: The architecture of P. fluorescens subsp. cellulosa has been determined crystallographically to be a commonly occurring enzyme fold, the eight-fold alpha/beta-barrel. Xylopentaose binds across the carboxy-terminal end of the alpha/beta-barrel in an active-site cleft which contains the two catalytic glutamates.

Insulin-stimulated GLUT4 glucose transporter recycling. A problem in membrane protein subcellular trafficking through multiple pools.

The subcellular trafficking of GLUT4 in isolated rat adipose cells and 3T3-L1 adipocytes exhibits many of the properties observed in regulated secretory processes and neurosecretion. GLUT4 is sorted and sequestered from endosomes into a specialized secretory compartment in the basal state and the initial stimulation of its exocytosis by insulin is more rapid than its recycling through the endosomes and secretory compartment during the steady-state response to insulin. We present a mathematical analysis which shows that this behavior is inconsistent with a simple 2-pool model with one plasma membrane and one intracellular compartment, but that a 3-pool model, with two intracellular compartments, can simulate these properties. We extend this model to include the presence of occluded pools in the plasma membrane. Our analysis compares the behavior expected when these occluded pools are precursors in stimulation and/or clathrin-associated-like intermediates in endocytosis. The presence of a precursor occluded pool can account for a lag between the appearance of GLUT4 in the membrane and before the full stimulation of glucose transport activity. The analysis also shows that since the pool size of the occluded GLUT4 is relatively small, the formation of endocytic occluded intermediates such as GLUT4 in clathrin-coated pits is likely to be slow compared with the rate of endocytosis of the coated vesicles.

Some effects of zona pellucida glycoproteins and sulfated polymers on the autoactivation of boar sperm proacrosin and activity of beta-acrosin.

The effects of zona pellucida glycoproteins, sulfated polymers and non-sulfated polymers on the activation kinetics of boar sperm proacrosin to beta-acrosin have been investigated. The aim has been to understand more about the behaviour and function of this protein after it has been released from the acrosome at the time of fertilization. Purified proacrosin was allowed to autoactivate at pH 8.0 in the presence of different concentrations of homologous zona glycoproteins, sulfated polymers (fucoidan, chondroitin sulfates A, B and C, dextran sulfate, polyvinylsulfate and heparin) and non-sulfated polymers (dextran, polyvinylphosphate and hyaluronic acid). Enzyme activity was measured against N-benzoyl-L-arginine p-nitroanalide substrate and changes in molecular mass of the protein monitored by SDS-PAGE. Results show that zona pellucida glycoproteins, fucoidan, dextran sulfate and polyvinylsulfate all potentiate the conversion of proacrosin to beta-acrosin but subsequently inhibit its amidase activity. Dextran, polyvinylphosphate, chondroitin sulfates A, B and C and glucose-6-sulfate, on the other hand, either have no effect on autoactivation and beta-acrosin activity, or enhance it slightly. SDS-PAGE analysis confirmed these observations and further indicated that binding of sulfated polymers to proacrosin inhibited staining by Coomassie Blue. These results are consistent with the hypothesis that binding of zona pellucida glycoproteins and sulfated compounds to proacrosin/acrosin is stereospecific and that contact activation onto soluble 'surfaces' causes conformational changes that are responsible for potentiation or inhibition of activation. The implications of these findings for sperm binding and penetration of the zona pellucida are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)