The glucanases of Cellulomonas

Cellulomonas is a unique bacterium possessing not only the capacity to degrade various carbohydrates, such as starch, xylan and cellulose, but crystalline cellulose as well. It has developed a complex battery of glucanases to deal with substrates possessing such extensive microheterogeneities. Some of these enzymes are multifunctional, as well as cross inducible, possessing a multi-domain structure; these enzymes are thought to have arisen by the shuffling of these domains. Intergeneric hybrids have been constructed between Cellulomonas and Zymomonas so as to enhance the industrial potential of this organism. This review examines the unique features of this microorganism and evaluates its key role in the conversion of complex wastes to useful products, by virtue of its unusual attributes.     

TPR-containing proteins control protein organization and homeostasis for the endoplasmic reticulum

The endoplasmic reticulum (ER) is a complex, multifunctional organelle comprised of a continuous membrane and lumen that is organized into a number of functional regions. It plays various roles including protein translocation, folding, quality control, secretion, calcium signaling, and lipid biogenesis. Cellular protein homeostasis is maintained by a complicated chaperone network, and the largest functional family within this network consists of proteins containing tetratricopeptide repeats (TPRs). TPRs are well-studied structural motifs that mediate intermolecular protein–protein interactions, supporting interactions with a wide range of ligands or substrates. Seven TPR-containing proteins have thus far been shown to localize to the ER and control protein organization and homeostasis within this multifunctional organelle. Here, we discuss the roles of these proteins in controlling ER processes and organization. The crucial roles that TPR-containing proteins play in the ER are highlighted by diseases or defects associated with their mutation or disruption.     

Clathrin-associated adaptor proteins - putting it all together

Adaptors are multifunctional linker proteins that, as a coated vesicle assembles, tether the clathrin lattice to the underlying membrane bud site. Each adaptor is composed of four distinct protein submits, but how these assemble into the functional complex is not clear. Here, some features of the protein sequences are discussed in an attempt to develop a speculative, low-resolution structural model of possible subunit interactions.     

An in vitro enzymatic assay coupled to proteomics analysis reveals a new DNA processing activity for Ewing sarcoma and TAF(II)68 proteins

Based on structural and functional similarities, translocated in liposarcoma/fusion (TLS/FUS) protein, Ewing sarcoma (EWS) protein and human TATA binding protein-associated factor (hTAF(II)68) have been grouped in the TLS-EWS-TAF(II)68 (TET) protein family. Translocations involving their genes lead to sarcomas. Polypyrimidine tract-binding protein-associated splicing factor (PSF), although not grouped in this family, presents structural and functional similarities with TET proteins and is involved in translocation leading to carcinoma. Beside their role in RNA metabolism, the precise cellular functions of these multifunctional proteins are not yet fully elucidated. We previously showed that both TLS/FUS and PSF display activities able to pair homologous DNA on membrane in an in vitro assay. In the present study, we address the question whether EWS and hTAF(II)68 also display pairing on membrane activities, and to a larger extent whether other proteins also exhibit such activity. We applied the pairing on membrane assay to 2-DE coupled to MS analysis for a global screening of DNA pairing on membrane activities. In addition to TLS/FUS and PSF, this test allowed us to identify EWS and hTAF(II)68, but no other proteins, indicating a feature specific to a protein family whose members share extensive structural similarities. This common activity suggests a role for TET proteins and PSF in genome plasticity control.     

The Active Site of a Carbohydrate Esterase Displays Divergent Catalytic and Noncatalytic Binding Functions

Multifunctional proteins, which play a critical role in many biological processes, have typically evolved through the recruitment of different domains that have the required functional diversity. Thus the different activities displayed by these proteins are mediated by spatially distinct domains, consistent with the specific chemical requirements of each activity. Indeed, current evolutionary theory argues that the colocalization of diverse activities within an enzyme is likely to be a rare event, because it would compromise the existing activity of the protein. In contrast to this view, a potential example of multifunctional recruitment into a single protein domain is provided by CtCel5C-CE2, which contains an N-terminal module that displays cellulase activity and a C-terminal module, CtCE2, which exhibits a noncatalytic cellulose-binding function but also shares sequence identity with the CE2 family of esterases. Here we show that, unlike other CE2 members, the CtCE2 domain displays divergent catalytic esterase and noncatalytic carbohydrate binding functions. Intriguingly, these diverse activities are housed within the same site on the protein. Thus, a critical component of the active site of CtCE2, the catalytic Ser-His dyad, in harness with inserted aromatic residues, confers noncatalytic binding to cellulose whilst the active site of the domain retains its esterase activity. CtCE2 catalyses deacetylation of noncellulosic plant structural polysaccharides to deprotect these substrates for attack by other enzymes. Yet it also acts as a cellulose-binding domain, which promotes the activity of the appended cellulase on recalcitrant substrates. The CE2 family encapsulates the requirement for multiple activities by biocatalysts that attack challenging macromolecular substrates, including the grafting of a second, powerful and discrete noncatalytic binding functionality into the active site of an enzyme. This article provides a rare example of “gene sharing,” where the introduction of a second functionality into the active site of an enzyme does not compromise the original activity of the biocatalyst.     

The multifaceted roles of the HORMA domain in cellular signaling

The HORMA domain is a multifunctional protein–protein interaction module found in diverse eukaryotic signaling pathways including the spindle assembly checkpoint, numerous DNA recombination/repair pathways, and the initiation of autophagy. In all of these pathways, HORMA domain proteins occupy key signaling junctures and function through the controlled assembly and disassembly of signaling complexes using a stereotypical “safety belt” peptide interaction mechanism. A recent explosion of structural and functional work has shed new light on these proteins, illustrating how strikingly similar structural mechanisms give rise to radically different functional outcomes in each family of HORMA domain proteins.     

Crystallographic studies on human BST-1/CD157 with ADP-ribosyl cyclase and NAD glycohydrolase activities

cADPR is the novel second messenger that elicits calcium release from intracellular calcium stores and works independently of IP(3). In mammals, the ADP-ribosyl cyclase function is found in two membrane proteins, CD38 and BST-1/CD157. These enzymes, exposed extracellularly, bear cADPR hydrolase and NAD glycohydrolase activities. In spite of its functional importance, the structural basis of these enzymatic reactions remains elusive. We determined the crystal structures of the extracellular region of human BST-1 at atomic resolution in the free form and in complexes with five substrate analogues: nicotinamide, NMN, ATPgammaS, ethenoNADP, and ethenoNAD. The three-dimensional structural views of the reaction centre with these ligands revealed the mode of substrate binding and the catalytic mechanism of the multifunctional enzymatic reactions. In each catalytic cleft of the dimeric enzyme, substrates are recognized predominantly through van der Waals interactions with two tryptophan residues, and thereby the N-glycosidic bond of NAD is correctly exposed near a catalytic glutamate residue. Its carboxyl side-chain stabilizes the catalytic intermediate of the S(N)-1 type reaction. This conformation of the catalytic cleft also implies the mechanism of cyclization between the adenine base and the ribose. The three key residues are invariant among the sequences of BST-1, CD38, and Aplysia cyclase, and hence this substrate recognition mode and catalytic scheme appear to be common in the cyclase family.     

Characteristics of Pathogenesis-Related Proteins Induced in Phaseolus vulgaris cv. Pinto Following Viral Infection

Ten acidic and eight basic pathogenesis-related (PR) proteins were induced in primary leaves of Phaseolus vulgaris cv. Pinto following infection with southern bean mosaic virus (SBMV), a virus that induces circular, brown necrotic local lesions. These PR proteins included: four, 17 kDa, serologically related, acidic proteins of unknown functions; two chitinases, one acidic (29 kDa) and one basic (32 kDa) possessing antifungal activities; and four (21 kDa, 28 kDa, 29 kDa, 36 kDa) serologically related, acidic glucanases. The production of PR proteins was minimal during the period of active SBMV multiplication; greatest increase of PR proteins occurred after SBMV had reached a maximum level. Several of these PR proteins, including the 29 kDa acidic chitinase and two acidic glucanases (21 kDa and 36 kDa) accumulated in the intercellular space fluid. Similar PR proteins were synthesized in Pinto bean primary leaves infects with any of three other viruses that formed lesions of divergent phenotypes. However, the proportion of some of the PR proteins in the various lesion-types differed considerably. These results indicate that PR proteins play no active role in viral localization or inactivation and that their production is a metabolic adjustment to endogenous stress.     

Glucanase gene diversity in prokaryotic and eukaryotic organisms

A number of bacteria and eukaryotes produce extracellular enzymes that degrade various types of polysaccharides including the glucans starch, cellulose and hemicellulose (xylan). The similarities in the modes of expression and specificity of enzyme classes, such as amylase, cellulose and xylanase, suggest common genetic origins for particular activities. Our determination of the extent of similarity between these glucanases suggests that such data may be of very limited use in describing the early evolution of these proteins. The great diversity of these proteins does allow identification of their most highly conserved (and presumably functionally important) regions.     

Antifungal proteins and peptides of leguminous and non-leguminous origins

Antifungal proteins and peptides, as their names imply, serve a protective function against fungal invasion. They are produced by a multitude of organisms including leguminous flowering plants, non-leguminous flowering plants, gymnosperms, fungi, bacteria, insects and mammals. The intent of the present review is to focus on the structural and functional characteristics of leguminous, as well as non-leguminous, antifungal proteins and peptides. A spectacular diversity of amino acid sequences has been reported. Some of the antifungal proteins and peptides are classified, based on their structures and/or functions, into groups including chitinases, glucanases, thaumatin-like proteins, thionins, and cyclophilin-like proteins. Some of the well-known proteins such as lectins, ribosome inactivating proteins, ribonucleases, deoxyribonucleases, peroxidases, and protease inhibitors exhibit antifungal activity. Different antifungal proteins may demonstrate different fungal specificities. The mechanisms of antifungal action of only some antifungal proteins including thaumatin-like proteins and chitinases have been elucidated.     

Purification of the fengycin synthetase multienzyme system from Bacillus subtilis b213

The purification of the multienzyme system producing the lipodecapeptide fengycin in Bacillus subtilis b213 was investigated. By gel filtration of a cell free extract of this organism three enzyme fractions were obtained from which five multifunctional components of fengycin synthetase were separated by high resolution anion-exchange FPLC procedures. These proteins were characterized by their thioester formation activities with ¹⁴C-labeled substrate amino acids and by N-terminal sequencing. Correlation of these data with the DNA sequences of the pps (fen) operons in three B. subtilis strains provided detailed knowledge on the structural and functional organization of fengycin synthetase.     

A-KinaseAnchoringProteins: a key to selective activation of cAMP-responsive events?

The cAMP-dependent protein kinase (PKA) regulates a variety of diverse biochemical events through the phosphorylation of target proteins. Because PKA is a multifunctional enzyme with a broad substrate specificity, its compartmentalization may be a key regulatory event in controlling which particular target substrates are phosphorylated. In recent years it has been demonstrated that differential localization of the type II holoenzyme is directed through interaction of the regulatory subunit (RII) with a family ofA-KinaseAnchoringProteins (AKAPs). In this report, we review evidence for PKA compartmentalization and discuss the structural and functional properties of AKAPs.     

Multiple Horizontal Gene Transfer Events and Domain Fusions Have Created Novel Regulatory and Metabolic Networks in the Oomycete Genome

Complex enzymes with multiple catalytic activities are hypothesized to have evolved from more primitive precursors. Global analysis of the Phytophthora sojae genome using conservative criteria for evaluation of complex proteins identified 273 novel multifunctional proteins that were also conserved in P. ramorum. Each of these proteins contains combinations of protein motifs that are not present in bacterial, plant, animal, or fungal genomes. A subset of these proteins were also identified in the two diatom genomes, but the majority of these proteins have formed after the split between diatoms and oomycetes. Documentation of multiple cases of domain fusions that are common to both oomycetes and diatom genomes lends additional support for the hypothesis that oomycetes and diatoms are monophyletic. Bifunctional proteins that catalyze two steps in a metabolic pathway can be used to infer the interaction of orthologous proteins that exist as separate entities in other genomes. We postulated that the novel multifunctional proteins of oomycetes could function as potential Rosetta Stones to identify interacting proteins of conserved metabolic and regulatory networks in other eukaryotic genomes. However ortholog analysis of each domain within our set of 273 multifunctional proteins against 39 sequenced bacterial and eukaryotic genomes, identified only 18 candidate Rosetta Stone proteins. Thus the majority of multifunctional proteins are not Rosetta Stones, but they may nonetheless be useful in identifying novel metabolic and regulatory networks in oomycetes. Phylogenetic analysis of all the enzymes in three pathways with one or more novel multifunctional proteins was conducted to determine the probable origins of individual enzymes. These analyses revealed multiple examples of horizontal transfer from both bacterial genomes and the photosynthetic endosymbiont in the ancestral genome of Stramenopiles. The complexity of the phylogenetic origins of these metabolic pathways and the paucity of Rosetta Stones relative to the total number of multifunctional proteins suggests that the proteome of oomycetes has few features in common with other Kingdoms.     

Cold-active winter rye glucanases with ice-binding capacity

Extracellular pathogenesis-related proteins, including glucanases, are expressed at cold temperatures in winter rye (Secale cereale) and display antifreeze activity. We have characterized recombinant cold-induced glucanases from winter rye to further examine their roles and contributions to cold tolerance. Both basic beta-1,3-glucanases and an acidic beta-1,3;1,4-glucanase were expressed in Escherichia coli, purified, and assayed for their hydrolytic and antifreeze activities in vitro. All were found to be cold active and to retain partial hydrolytic activity at subzero temperatures (e.g. 14%-35% at -4 degrees C). The two types of glucanases had antifreeze activity as measured by their ability to modify the growth of ice crystals. Structural models for the winter rye beta-1,3-glucanases were developed on which putative ice-binding surfaces (IBSs) were identified. Residues on the putative IBSs were charge conserved for each of the expressed glucanases, with the exception of one beta-1,3-glucanase recovered from nonacclimated winter rye in which a charged amino acid was present on the putative IBS. This protein also had a reduced antifreeze activity relative to the other expressed glucanases. These results support the hypothesis that winter rye glucanases have evolved to inhibit the formation of large, potentially fatal ice crystals, in addition to having enzymatic activity with a potential role in resisting infection by psychrophilic pathogens. Glucanases of winter rye provide an interesting example of protein evolution and adaptation aimed to combat cold and freezing conditions.     

Emerging roles of ADAM and ADAMTS metalloproteinases in cancer

A disintegrin and metalloproteinases (ADAMs) are a recently discovered family of proteins that share the metalloproteinase domain with matrix metalloproteinases (MMPs). Among this family, structural features distinguish the membrane-anchored ADAMs and the secreted ADAMs with thrombospondin motifs referred to as ADAMTSs. By acting on a large panel of membrane-associated and extracellular substrates, they control several cell functions such as adhesion, fusion, migration and proliferation. The current review addresses the contribution of these proteinases in the positive and negative regulation of cancer progression as mainly mediated by the regulation of growth factor activities and integrin functions.     

Combining structural genomics and enzymology: completing the picture in metabolic pathways and enzyme active sites

An important goal of structural genomics is to complete the structural analysis of all the enzymes in metabolic pathways and to understand the structural similarities and differences. A preliminary glimpse of this type of analysis was achieved before structural genomics efforts with the glycolytic pathway and efforts are underway for many other pathways, including that of catecholamine metabolism. Structural enzymology necessitates a complete structural characterization, even for highly homologous proteins (greater than 80% sequence homology), as every active site has distinct structural features and it is these active site differences that distinguish one enzyme from another. Short cuts with homology modeling cannot be taken with our current knowledge base. Each enzyme structure in a pathway needs to be determined, including structures containing bound substrates, cofactors, products and transition state analogs, in order to obtain a complete structural and functional understanding of pathway-related enzymes.