Structural and Functional Comparison of Polysaccharide-Degrading Enzymes

Sugar molecules as well as enzymes degrading them are ubiquitously present in physiological systems, especially for vertebrates. Polysaccharides have at least two aspects to their function, one due to their mechanical properties and the second one involves multiple regulatory processes or interactions between molecules, cells, or extracellular space. Various bacteria exert exogenous pressures on their host organism to diversity glycans and their structures in order for the host organism to evade the destructive function of such microbes. Many bacterial organism produce glycan-degrading enzymes in order to facilitate their invasion of host tissues. Such polysaccharide degrading enzymes utilize mainly two modes of polysaccharide-degradation, a hydrolysis and a β-elimination process. The three-dimensional structures of several of these enzymes have been elucidated recently using X-ray crystallography. There are many common structural motifs among these enzymes, mainly the presence of an elongated cleft transversing these molecules which functions as a polysaccharide substrate binding site as well as the catalytic site for these enzymes. The detailed structural information obtained about these enzymes allowed formulation of proposed mechanisms of their action. The polysaccharide lyases utilize a proton acceptance and donation mechanism (PAD), whereas polysaccharide hydrolases use a direct double displacement (DD) mechanism to degrade their substrates.     

Structural and functional comparison of polysaccharide-degrading enzymes

Sugar molecules as well as enzymes degrading them are ubiquitously present in physiological systems, especially for vertebrates. Polysaccharides have at least two aspects to their function, one due to their mechanical properties and the second one involves multiple regulatory processes or interactions between molecules, cells, or extracellular space. Various bacteria exert exogenous pressures on their host organism to diversity glycans and their structures in order for the host organism to evade the destructive function of such microbes. Many bacterial organism produce glycan-degrading enzymes in order to facilitate their invasion of host tissues. Such polysaccharide degrading enzymes utilize mainly two modes of polysaccharide-degradation, a hydrolysis and a beta-elimination process. The three-dimensional structures of several of these enzymes have been elucidated recently using X-ray crystallography. There are many common structural motifs among these enzymes, mainly the presence of an elongated cleft transversing these molecules which functions as a polysaccharide substrate binding site as well as the catalytic site for these enzymes. The detailed structural information obtained about these enzymes allowed formulation of proposed mechanisms of their action. The polysaccharide lyases utilize a proton acceptance and donation mechanism (PAD), whereas polysaccharide hydrolases use a direct double displacement (DD) mechanism to degrade their substrates.     

Quinone-dependent alcohol dehydrogenases and fad-dependent alcohol oxidases

This review considers quinone-dependent alcohol dehydrogenases and FAD-dependent alcohol oxidases, enzymes that are present in numerous methylotrophic eu- and prokaryotes and significantly differ in their primary and quaternary structure. The cofactors of the enzymes are bound to the protein polypeptide chain through ionic and hydrophobic interactions. Microorganisms containing these enzymes are described. Methods for purification of the enzymes, their physicochemical properties, and spatial structures are considered. The supposed mechanism of action and practical application of these enzymes as well as their producers are discussed.     

The pharmacological role of nucleotidases in snake venoms

Several hydrolytic enzymes of snake venom have evolved to interfere in various physiological processes, which are well defined. However, hydrolytic enzymes such as nucleotidases (5′nucleotidase, ATPase, and ADPase) are less studied and their pharmacological role in venoms is not clearly defined. Very few studies have shown the pharmacological importance of these endogenous purine release related enzymes in venoms. The near‐ubiquitous distribution of these enzymes in venoms, suggests a significant role for these enzymes in envenomation. It is suggested that their major function is in the generation of purines (mainly adenosine)—a multitoxin. Therefore, it appears that these enzymes play a central role in liberating adenosine and through the action of adenosine help in prey immobilization. However, apart from this, these enzymes could also possess other pharmacological activities. Further research is needed to biologically characterize these enzymes in snake venoms, such that their role in venom is clearly established. Copyright © 2010 John Wiley & Sons, Ltd.     

α-l-Arabinofuranosidases: the potential applications in biotechnology

Recently, alpha-L-arabinofuranosidases (EC3.2.1.55) have received increased attention primarily due to their role in the degradation of lignocelluloses as well as their positive effect on the activity of other enzymes acting on lignocelluloses. As a result, these enzymes are used in many biotechnological applications including wine industry, clarification of fruit juices, digestion enhancement of animal feedstuffs and as a natural improver for bread. Moreover, these enzymes could be used to improve existing technologies and to develop new technologies. The production, mechanisms of action, classification, synergistic role, biochemical properties, substrate specificities, molecular biology and biotechnological applications of these enzymes have been reviewed in this article.     

Aminohydrolases acting on adenine, adenosine and their derivatives

BACKGROUND: Adenine and adenosine-acting aminohydrolases are important groups of enzymes responsible for the metabolic salvage of purine compounds. Several subclasses of these enzymes have been described and given current knowledge of the full genome sequences of many organisms, it is possible to identify genes encoding these enzymes and group them according to their primary structure. METHODS AND RESULTS: This article is a short overview of the enzymes classified as adenine and adenosine deaminase. It summarises knowledge of their occurrence, genetic basis and their catalytic and structural properties. CONCLUSIONS: These enzymes are constitutive components of purine metabolism and their impairment may cause serious medical disorders. In humans, adenosine deaminase deficiency is linked to severe combined immunodeficiency and as such the enzyme has been approved for the first gene therapy trial. The role of these enzymes in plants is unclear, since the activity was has not been detected in extracts and putative genes have not been yet cloned and analyzed. A literature search and amino acid identity comparison show that Ascomycetes contain only adenine deaminase, but not adenosine deaminase, despite the fact that corresponding genes are annotated in databases as the adenosine cleaving enzymes because they share the same conserved domain.     

The Mycobacterium tuberculosis FAS-II condensing enzymes: their role in mycolic acid biosynthesis, acid-fastness, pathogenesis and in future drug development

Mycolic acids are very long-chain fatty acids representing essential components of the mycobacterial cell wall. Considering their importance, characterization of key enzymes participating in mycolic acid biosynthesis not only allows an understanding of their role in the physiology of mycobacteria, but also might lead to the identification of new drug targets. Mycolates are synthesized by at least two discrete elongation systems, the type I and type II fatty acid synthases (FAS-I and FAS-II respectively). Among the FAS-II components, the condensing enzymes that catalyse the formation of carbon-carbon bonds have received considerable interest. Four condensases participate in initiation (mtFabH), elongation (KasA and KasB) and termination (Pks13) steps, leading to full-length mycolates. We present the recent biochemical and structural data for these important enzymes. Special emphasis is given to their role in growth, intracellular survival, biofilm formation, as well as in the physiopathology of tuberculosis. Recent studies demonstrated that phosphorylation of these enzymes by mycobacterial kinases affects their activities. We propose here a model in which kinases that sense environmental changes can phosphorylate the condensing enzymes, thus representing a novel mechanism of regulating mycolic acid biosynthesis. Finally, we discuss the attractiveness of these enzymes as valid targets for future antituberculosis drug development.     

Expression and secretion of glycosylated heparin biosynthetic enzymes using Komagataella pastoris

Heparin, an anticoagulant drug, is biosynthesized in selected animal cells. The heparin biosynthetic enzymes mainly consist of sulfotransferases and all are integral transmembrane glycoproteins. These enzymes are generally produced in engineered Escherichia coli as without their transmembrane domains as non-glycosylated fusion proteins. In this study, we used the yeast, Komagataella pastoris, to prepare four sulfotransferases involved in heparin biosynthesis as glycoproteins. While the yields of these yeast-expressed enzymes were considerably lower than E. coli-expressed enzymes, these enzymes were secreted into the fermentation media simplifying their purification and were endotoxin free. The activities of these sulfotransferases, expressed as glycoproteins in yeast, were compared to the bacterially expressed proteins. The yeast-expressed sulfotransferase glycoproteins showed improved kinetic properties than the bacterially expressed proteins.

     

A genomic and functional inventory of deubiquitinating enzymes

Posttranslational modification of proteins by the small molecule ubiquitin is a key regulatory event, and the enzymes catalyzing these modifications have been the focus of many studies. Deubiquitinating enzymes, which mediate the removal and processing of ubiquitin, may be functionally as important but are less well understood. Here, we present an inventory of the deubiquitinating enzymes encoded in the human genome. In addition, we review the literature concerning these enzymes, with particular emphasis on their function, specificity, and the regulation of their activity.     

The compartmentalization and translocation of the sphingosine kinases: mechanisms and functions in cell signaling and sphingolipid metabolism

Members of the sphingosine kinase (SK) family of lipid signaling enzymes, comprising SK1 and SK2 in humans, are receiving considerable attention for their roles in a number of physiological and pathophysiological processes. The SKs are considered signaling enzymes based on their production of the potent lipid second messenger sphingosine-1-phosphate, which is the ligand for a family of five G-protein-linked receptors. Both SK1 and SK2 are intracellular enzymes and do not possess obvious membrane anchor domains within their primary sequences. The native substrates (sphingosine and dihydrosphingosine) are lipids, as are the corresponding products, and therefore would have a propensity to be membrane associated, suggesting that specific membrane localization of the SKs could affect both access to substrate and localized production of product. Here, we consider the emerging picture of the SKs as enzymes localized to specific intracellular sites, sometimes by agonist-dependent translocation, the mechanism targeting these enzymes to those sites, and the functional consequence of that localization. Not only is the signaling output of the SKs affected by subcellular localization, but the role of these enzymes as metabolic regulators of sphingolipid metabolism may be impacted as well.     

DyP-type peroxidases: a promising and versatile class of enzymes

DyP peroxidases comprise a novel superfamily of heme-containing peroxidases, which is unrelated to the superfamilies of plant and animal peroxidases. These enzymes have so far been identified in the genomes of fungi, bacteria, as well as archaea, although their physiological function is still unclear. DyPs are bifunctional enzymes displaying not only oxidative activity but also hydrolytic activity. Moreover, these enzymes are able to oxidize a variety of organic compounds of which some are poorly converted by established peroxidases, including dyes, β-carotene, and aromatic sulfides. Interestingly, accumulating evidence shows that microbial DyP peroxidases play a key role in the degradation of lignin. Owing to their unique properties, these enzymes are potentially interesting for a variety of biocatalytic applications. In this review, we deal with the biochemical and structural features of DyP-type peroxidases as well as their promising biotechnological potential.     

The compartmentalization and translocation of the sphingosine kinases: Mechanisms and functions in cell signaling and sphingolipid metabolism

Members of the sphingosine kinase (SK) family of lipid signaling enzymes, comprising SK1 and SK2 in humans, are receiving considerable attention for their roles in a number of physiological and pathophysiological processes. The SKs are considered signaling enzymes based on their production of the potent lipid second messenger sphingosine-1-phosphate, which is the ligand for a family of five G-protein-linked receptors. Both SK1 and SK2 are intracellular enzymes and do not possess obvious membrane anchor domains within their primary sequences. The native substrates (sphingosine and dihydrosphingosine) are lipids, as are the corresponding products, and therefore would have a propensity to be membrane associated, suggesting that specific membrane localization of the SKs could affect both access to substrate and localized production of product. Here, we consider the emerging picture of the SKs as enzymes localized to specific intracellular sites, sometimes by agonist-dependent translocation, the mechanism targeting these enzymes to those sites, and the functional consequence of that localization. Not only is the signaling output of the SKs affected by subcellular localization, but the role of these enzymes as metabolic regulators of sphingolipid metabolism may be impacted as well.     

Plant protein inhibitors of cell wall degrading enzymes

Plant cell walls, which consist mainly of polysaccharides (i.e. cellulose, hemicelluloses and pectins), play an important role in defending plants against pathogens. Most phytopathogenic microorganisms secrete an array of cell wall degrading enzymes (CWDEs) capable of depolymerizing the polysaccharides in the plant host wall. In response, plants have evolved a diverse battery of defence responses including protein inhibitors of these enzymes. These include inhibitors of pectin degrading enzymes such as polygalacturonases, pectinmethyl esterases and pectin lyases, and hemicellulose degrading enzymes such as endoxylanases and xyloglucan endoglucanases. The discovery of these plant inhibitors and the recent resolution of their three-dimensional structures, free or in complex with their target enzymes, provide new lines of evidence regarding their function and evolution in plant-pathogen interactions.     

Enzyme promiscuity – A light on the “darker” side of enzyme specificity

Abstract

Enzyme promiscuity can be defined as the capability of enzymes to catalyse side reaction in addition to its main reaction. The side reaction of an enzyme is termed as promiscuous or sometimes as the “darker” side of enzyme cross-reactivity/specificity. This unique property of enzyme allows organisms to adapt under varying environmental conditions. Promiscuous enzymes can modify their catalytic activities with altered substrates and can adjust their catalytic and kinetic mechanisms according to substrate properties. This group of enzymes evolved from ancestral proteins found in primitive organisms like archaea that survive under extreme environmental conditions. Such ancestral proteins possessed the potential to catalyse a wide range of reactions at low levels, hence create families or superfamilies of highly specialized enzymes. Further, some enzymes were identified which have non-catalytic functions in addition to their major catalytic activities. These enzymes are referred to as moonlighting enzymes. The study of these enzymes will provide important information regarding enzyme evolution and will help in optimizing protein engineering applications.     

Pictet–Spenglerases in alkaloid biosynthesis: Future applications in biocatalysis

Pictet–Spenglerases provide a key role in the biosynthesis of many biologically active alkaloids. There is increasing use of these biocatalysts as an alternative to traditional organic synthetic methods as they provide stereoselective and regioselective control under mild conditions. Products from these enzymes also contain privileged drug scaffolds (such as tetrahydroisoquinoline or β-carboline moieties), so there is interest in the characterization and use of these enzymes as versatile biocatalysts to synthesize analogs of the corresponding natural products for drug discovery. This review discusses all known Pictet–Spenglerase enzymes and their applications as biocatalysts.     

Age-dependent changes in the activities of ATPase and some pyridine nucleotide-linked enzymes in the chick testis.

The ontogeny of some of the enzymes connected with carbohydrate metabolism in the testis were studied in the White-Rock chicks. In the first place testicular growth in these chicks relate to their overall growth as measured by their body weights. ATPase and NAD+-dependent succinic dehydrogenase activities decreased both with advancing age and increasing testicular weight. However, these enzymes showed maximum activities at 17 and 28 weeks respectively. NAD+-linked isocitric dehydrogenase activity continually increased with increasing testicular weight and age. It is suggested that during spermatogenesis the activities of these enzymes are controlled by different developmental mechanisms.     

Cold-Adapted Enzymes from Marine Antarctic Microorganisms

The Antarctic marine environment is characterized by challenging conditions for the survival of native microorganisms. Indeed, next to the temperature effect represented by the Arrhenius law, the viscosity of the medium, which is also significantly enhanced by low temperatures, contributes to slow down reaction rates. This review analyses the different challenges and focuses on a key element of life at low temperatures: cold-adapted enzymes. The molecular characteristics of these enzymes are discussed as well as the adaptation strategies which can be inferred from the comparison of their properties and three-dimensional structures with those of their mesophilic counterparts. As these enzymes display a high specific activity at low and moderate temperatures associated with a relatively high thermosensitivity, the interest in these properties is discussed with regard to their current and possible applications in biotechnology.     

Role of sphingosine kinase localization in sphingolipid signaling

The sphingosine kinases, SK1 and SK2, produce the potent signaling lipid sphingosine-1-phosphate (S1P). These enzymes have garnered increasing interest for their roles in tumorigenesis, inflammation, vascular diseases, and immunity, as well as other functions. The sphingosine kinases are considered signaling enzymes by producing S1P, and their activity is acutely regulated by a variety of agonists. However, these enzymes are also key players in the control of sphingolipid metabolism. A variety of sphingolipids, such as sphingosine and the ceramides, are potent signaling molecules in their own right. The role of sphingosine kinases in regulating sphingolipid metabolism is potentially a critical aspect of their signaling function. A central aspect of signaling lipids is that their hydrophobic nature constrains them to membranes. Most enzymes of sphingolipid metabolism, including the enzymes that degrade S1P, are membrane enzymes. Therefore the localization of the sphingosine kinases and S1P is likely to be important in S1P signaling. Sphingosine kinase localization affects sphingolipid signaling in several ways. Translocation of SK1 to the plasma membrane promotes extracellular secretion of S1P. SK1 and SK2 localization to specific sites appears to direct S1P to intracellular protein effectors. SK localization also determines the access of these enzymes to their substrates. This may be an important mechanism for the regulation of ceramide biosynthesis by diverting dihydrosphingosine, a precursor in the ceramide biosynthetic pathway, from the de novo production of ceramide.     

Evolutionary relationships of adenylation domains in fungi

Non-ribosomal peptide synthetases (NRPSs) and NRPS-like enzymes are abundant in microbes as they are involved in the production of primary and secondary metabolites. In contrast to the well-studied NRPSs, known to produce non-ribosomal peptides, NRPS-like enzymes exhibit more diverse activities and their evolutionary relationships are unclear. Here, we present the first in-depth phylogenetic analysis of fungal NRPS-like A domains from functionally characterized pathways, and their relationships to characterized A domains found in fungal NRPSs. This study clearly differentiated amino acid reductases, including NRPSs, from CoA/AMP ligases, which could be divided into 10 distinct phylogenetic clades that reflect their conserved domain organization, substrate specificity and enzymatic activity. In particular, evolutionary relationships of adenylate forming reductases could be refined and explained the substrate specificity difference. Consistent with their phylogeny, the deduced amino acid code of A domains differentiated amino acid reductases from other enzymes. However, a diagnostic code was found for α-keto acid reductases and clade 7 CoA/AMP ligases only. Comparative genomics of loci containing these enzymes revealed that they can be independently recruited as tailoring genes in diverse secondary metabolite pathways. Based on these results, we propose a refined and clear phylogeny-based classification of A domain-containing enzymes, which will provide a robust framework for future functional analyses and engineering of these enzymes to produce new bioactive molecules.