Catalytic mechanisms, basic roles, and biotechnological and environmental significance of halogenating enzymes

The understanding of enzymatic incorporation of halogen atoms into organic molecules has increased during the last few years. Two novel types of halogenating enzymes, flavindependent halogenases and α-ketoglutarate-dependent halogenases, are now known to play a significant role in enzyme-catalyzed halogenation. The recent advances on the halogenating enzymes RebH, SyrB2, and CytC3 have suggested some new mechanisms for enzymatic halogenations. This review concentrates on the occurrence, catalytic mechanisms, and biotechnological applications of the halogenating enzymes that are currently known.     

Structural and biochemical studies of GH family 12 cellulases: improved thermal stability, and ligand complexes

In this review we will describe how we have gathered structural and biochemical information from several homologous cellulases from one class of glycoside hydrolases (GH family 12), and used this information within the framework of a protein-engineering program for the design of new variants of these enzymes. These variants have been characterized to identify some of the positions and the types of mutations in the enzymes that are responsible for some of the biochemical differences in thermal stability and activity between the homologous enzymes. In this process we have solved the three-dimensional structure of four of these homologous GH 12 cellulases: Three fungal enzymes, Humicola grisea Cel12A, Hypocrea jecorina Cel12A and Hypocrea schweinitzii Cel12A, and one bacterial, Streptomyces sp. 11AG8 Cel12A. We have also determined the three-dimensional structures of the two most stable H. jecorina Cel12A variants. In addition, four ligand-complex structures of the wild-type H. grisea Cel12A enzyme have been solved and have made it possible to characterize some of the interactions between substrate and enzyme. The structural and biochemical studies of these related GH 12 enzymes, and their variants, have provided insight on how specific residues contribute to protein thermal stability and enzyme activity. This knowledge can serve as a structural toolbox for the design of Cel12A enzymes with specific properties and features suited to existing or new applications.     

Structural and biochemical studies of GH family 12 cellulases: improved thermal stability,and ligand complexes

In this review we will describe how we have gathered structural and biochemical information from several homologous cellulases from one class of glycoside hydrolases (GH family 12), and used this information within the framework of a protein-engineering program for the design of new variants of these enzymes. These variants have been characterized to identify some of the positions and the types of mutations in the enzymes that are responsible for some of the biochemical differences in thermal stability and activity between the homologous enzymes. In this process we have solved the three-dimensional structure of four of these homologous GH 12 cellulases: Three fungal enzymes, Humicola grisea Cel12A, Hypocrea jecorina Cel12A and Hypocrea schweinitzii Cel12A, and one bacterial, Streptomyces sp. 11AG8 Cel12A. We have also determined the three-dimensional structures of the two most stable H. jecorina Cel12A variants. In addition, four ligand-complex structures of the wild-type H. grisea Cel12A enzyme have been solved and have made it possible to characterize some of the interactions between substrate and enzyme. The structural and biochemical studies of these related GH 12 enzymes, and their variants, have provided insight on how specific residues contribute to protein thermal stability and enzyme activity. This knowledge can serve as a structural toolbox for the design of Cel12A enzymes with specific properties and features suited to existing or new applications.     

Multifunctional enzymes in archaea: promiscuity and moonlight

Enzymes from many archaea colonizing extreme environments are of great interest because of their potential for various biotechnological processes and scientific value of evolution. Many enzymes from archaea have been reported to catalyze promiscuous reactions or moonlight in different functions. Here, we summarize known archaeal enzymes of both groups that include different kinds of proteins. Knowledge of their biochemical properties and three-dimensional structures has proved invaluable in understanding mechanism, application, and evolutionary implications of this manifestation. In addition, the review also summarizes the methods to unravel the extra function which almost was discovered serendipitously. The study of these amazing enzymes will provide clues to optimize protein engineering applications and how enzymes might have evolved on Earth.     

Molecular Dynamics of Thermoenzymes at High Temperature and Pressure: A Review

Lipases are known for their versatility in addition to their ability to digest fat. They can be used for the formulation of detergents, as food ingredients and as biocatalysts in many industrial processes. Because conventional enzymes are frangible at high temperatures, the replacement of conventional chemical routes with biochemical processes that utilize thermostable lipases is vital in the industrial setting. Recent theoretical studies on enzymes have provided numerous fundamental insights into the structures, folding mechanisms and stabilities of these proteins. The studies corroborate the experimental results and provide additional information regarding the structures that were determined experimentally. In this paper, we review the computational studies that have described how temperature affects the structure and dynamics of thermoenzymes, including the thermoalkalophilic L1 lipase derived from Bacillus stearothermophilus. We will also discuss the potential of using pressure for the analysis of the stability of thermoenzymes because high pressure is also important for the processing and preservation of foods.     

Microbial biosynthesis of halometabolites

Halometabolites are compounds that are commonly found in nature and they are produced by many different organisms. Whereas bromometabolites can mainly be found in the marine environment, chlorometabolites are predominately produced by terrestrial organisms; iodo- and fluorocompounds are only produced infrequently. The halogen atoms are incorporated into organic compounds by enzyme-catalyzed reactions with halide ions as the halogen source. For over 40 years haloperoxidases were thought to be responsible for the incorporation of halogen atoms into organic molecules. However, haloperoxidases lack substrate specificity and regioselectivity, and the connection of haloperoxidases with the in vivo formation of halometabolites has never been demonstrated. Recently, molecular genetic investigations showed that, at least in bacteria, a different class of halogenases is involved in halometabolite formation. These halogenases were found to require FADH2, which can be produced from FAD and NADH by unspecific flavin reductases. In addition to FADH2, oxygen and halide ions (chloride and bromide) are necessary for the halogenation reaction. The FADH2-dependent halogenases show substrate specificity and regioselectivity, and their genes have been detected in many halometabolite-producing bacteria, suggesting that this type of halogenating enzymes constitutes the major source for halometabolite formation in bacteria and possibly also in other organisms.     

Structural and functional dissection of aminocoumarin antibiotic biosynthesis: a review

Aminocoumarin antibiotics are natural products of soil-dwelling bacteria called Streptomycetes. They are potent inhibitors of DNA gyrase, an essential bacterial enzyme and validated drug target, and thus have attracted considerable interest as potential templates for drug development. To date, aminocoumarins have not seen widespread clinical application on account of their poor pharmacological properties. Through studying the structures and mechanisms of enzymes from their biosynthetic pathways we will be better informed to redesign these compounds through rational pathway engineering. Novobiocin, the simplest compound, requires at least seventeen gene products to convert primary metabolites into the mature antibiotic. We have solved the crystal structures of four diverse biosynthetic enzymes from the novobiocin pathway, and used these as three-dimensional frameworks for the interpretation of functional and mechanistic data, and to speculate about how they might have evolved. The structure determinations have ranged from the routine to the challenging, necessitating a variety of different approaches.     

Actin binding proteins

In order to metastasize away from the primary tumor site and migrate into adjacent tissues, cancer cells will stimulate cellular motility through the regulation of their cytoskeletal structures. Through the coordinated polymerization of actin filaments, these cells will control the geometry of distinct structures, namely lamella, lamellipodia and filopodia, as well as the more recently characterized invadopodia. Because actin binding proteins play fundamental functions in regulating the dynamics of actin polymerization, they have been at the forefront of cancer research. This review focuses on a subset of actin binding proteins involved in the regulation of these cellular structures and protrusions, and presents some general principles summarizing how these proteins may remodel the structure of actin. The main body of this review aims to provide new insights into how the expression of these actin binding proteins is regulated during carcinogenesis and highlights new mechanisms that may be initiated by the metastatic cells to induce aberrant expression of such proteins.     

ACE for all – a molecular perspective

Angiotensin-I converting enzyme (ACE, EC 3.4.15.1) is a zinc dependent dipeptidyl carboxypeptidase with an essential role in mammalian blood pressure regulation as part of the renin-angiotensin aldosterone system (RAAS). As such, it has long been targeted in the treatment of hypertension through the use of ACE inhibitors. Although ACE has been studied since the 1950s, only recently have the full range of functions of this enzyme begun to truly be appreciated. ACE homologues have been found in a host of other organisms, and are now known to be conserved in insects. Insect ACE homologues typically share over 30 % amino acid sequence identity with human ACE. Given that insects lack a mammalian type circulatory system, they must have crucial roles in other physiological processes. The first ACE crystal structures were reported during the last decade and have enabled these enzymes to be studied from an entirely different perspective. Here we review many of these key developments and the implications that they have had on our understanding of the diverse functions of these enzymes. Specifically, we consider how structural information is being used in the design of a new generation of ACE inhibitors with increased specificity, and how the structures of ACE homologues are related to their functions. The Anopheles gambiae genome is predicted to code for ten ACE homologues, more than any genome studied so far. We have modelled the active sites of some of these as yet uncharacterised enzymes to try and infer more about their potential roles at the molecular level.     

Limb regeneration in axolotl: is it superhealing?

The ability of axolotls to regenerate their limbs is almost legendary. In fact, urodeles such as the axolotl are the only vertebrates that can regenerate multiple structures like their limbs, jaws, tail, spinal cord, and skin (the list goes on) throughout their lives. It is therefore surprising to realize, although we have known of their regenerative potential for over 200 years, how little we understand the mechanisms behind this achievement of adult tissue morphogenesis. Many observations can be drawn between regeneration and other disciplines such as development and wound healing. In this review, we present new developments in functional analysis that will help to address the role of specific genes during the process of regeneration. We also present an analysis of the resemblance between wound healing and regeneration, and discuss whether axolotls are superhealers. A better understanding of these animals' regenerative capacity could lead to major benefits by providing regenerative medicine with directions on how to develop therapeutic approaches leading to regeneration in humans.     

Structure and Action of the Myxobacterial Chondrochloren Halogenase CndH: A New Variant of FAD-dependent Halogenases

The crystal structure of the FAD-dependent chondrochloren halogenase CndH has been established at 2.1 Å resolution. The enzyme contains the characteristic FAD-binding scaffold of the glutathione reductase superfamily. Except for its C-terminal domain, the chainfold of CndH is virtually identical with those of FAD-dependent aromatic hydroxylases. When compared to the structurally known FAD-dependent halogenases PrnA and RebH, CndH lacks a 45 residue segment near position 100 and deviates in the C-terminal domain. Both variations are near the active center and appear to reflect substrate differences. Whereas PrnA and RebH modify free tryptophan, CndH halogenates the tyrosyl group of a chondrochloren precursor that is most likely bound to a carrier protein. In contrast to PrnA and RebH, which enclose their small substrate completely, CndH has a large non-polar surface patch that may accommodate the putative carrier. Apart from the substrate binding site, the active center of CndH corresponds to those of PrnA and RebH. At the halogenation site, CndH has the characteristic lysine (Lys76) but lacks the required base Glu346 (PrnA). This base may be supplied by a residue of its C-terminal domain or by the carrier. These differences were corroborated by an overall sequence comparison between the known FAD-dependent halogenases, which revealed a split into a PrnA-RebH group and a CndH group. The two functionally established members of the CndH group use carrier-bound substrates, whereas three members of PrnA-RebH group are known to accept a free amino acid. Given the structural and functional distinction, we classify CndH as a new variant B of the FAD-dependent halogenases, adding a new feature to the structurally established variant A enzymes PrnA and RebH.     

Flavin-dependent halogenases involved in secondary metabolism in bacteria

The understanding of biological halogenation has increased during the last few years. While haloperoxidases were the only halogenating enzymes known until 1997, it is now clear that haloperoxidases are hardly, if at all, involved in biosynthesis of more complex halogenated compounds in microorganisms. A novel type of halogenating enzymes, flavin-dependent halogenases, has been identified as a major player in the introduction of chloride and bromide into activated organic molecules. Flavin-dependent halogenases require the activity of a flavin reductase for the production of reduced flavin, required by the actual halogenase. A number of flavin-dependent tryptophan halogenases have been investigated in some detail, and the first three-dimensional structure of a member of this enzyme subfamily, tryptophan 7-halogenase, has been elucidated. This structure suggests a mechanism involving the formation of hypohalous acid, which is used inside the enzyme for regioselective halogenation of the respective substrate. The introduction of halogen atoms into non-activated alkyl groups is catalysed by non-heme Fe^II α-ketoglutarate- and O₂-dependent halogenases. Examples for the use of flavin-dependent halogenases for the formation of novel halogenated compounds in in vitro and in vivo reactions promise a bright future for the application of biological halogenation reactions.     

Flavin-containing heme enzymes

There are many examples of oxidative enzymes containing both flavin and heme prosthetic groups that carry out the oxidation of their substrate. For the purpose of this article we have chosen five systems. Two of these, the l-lactate dehydrogenase flavocytochrome b₂ and cellobiose dehydrogenase, carry out the catalytic chemistry at the flavin group. In contrast, the remaining three require activation of dioxygen at the heme group in order to accomplish substrate oxidation, these being flavohemoglobin, a nitric oxide dioxygenase, and the mono-oxygenases nitric oxide synthase and flavocytochrome P450 BM3, which functions as a fatty acid hydroxylase. In the light of recent advances we will describe the structures of these enzymes, some of which share significant homology. We will also discuss their diverse and sometimes controversial catalytic mechanisms, and consider electron transfer processes between the redox cofactors in order to provide an overview of this fascinating set of enzymes.     

Presynaptic neurotoxins: An expanding array of natural and modified molecules

The process of neurotransmitter release from nerve terminals is a target for a wide array of presynaptic toxins produced by various species, from humble bacteria to arthropods to vertebrate animals. Unlike other toxins, most presynaptic neurotoxins do not kill cells but simply inhibit or activate synaptic transmission. In this review, we describe two types of presynaptic neurotoxins: clostridial toxins and latrotoxins, which are, respectively, the most potent blockers and stimulators of neurotransmitter release. These toxins have been instrumental in defining presynaptic functions and are now widely used in research and medicine. Here, we would like to analyse the diversity of these toxins and demonstrate how the knowledge of their structures and mechanisms of action can help us to design better tools for research and medical applications. We will look at natural and synthetic variations of these exquisite molecular machines, highlighting recent advances in our understanding of presynaptic toxins and questions that remain to be answered. If we can decipher how a given biomolecule is modified by nature to target different species, we will be able to design new variants that carry only desired characteristics to achieve specific therapeutic, agricultural or research goals. Indeed, a number of research groups have already initiated a quest to harness the power of natural toxins with the aim of making them more specifically targeted and safer for future research and medical applications.     

New Insights about Enzyme Evolution from Large Scale Studies of Sequence and Structure Relationships

Understanding how enzymes have evolved offers clues about their structure-function relationships and mechanisms. Here, we describe evolution of functionally diverse enzyme superfamilies, each representing a large set of sequences that evolved from a common ancestor and that retain conserved features of their structures and active sites. Using several examples, we describe the different structural strategies nature has used to evolve new reaction and substrate specificities in each unique superfamily. The results provide insight about enzyme evolution that is not easily obtained from studies of one or only a few enzymes.     

Perspective: a stirring role for metabolism in cells

Based on recent findings indicating that metabolism might be governed by a limit on the rate at which cells can dissipate Gibbs energy, in this Perspective, we propose a new mechanism of how metabolic activity could globally regulate biomolecular processes in a cell. Specifically, we postulate that Gibbs energy released in metabolic reactions is used to perform work, allowing enzymes to self‐propel or to break free from supramolecular structures. This catalysis‐induced enzyme movement will result in increased intracellular motion, which in turn can compromise biomolecular functions. Once the increased intracellular motion has a detrimental effect on regulatory mechanisms, this will establish a feedback mechanism on metabolic activity, and result in the observed thermodynamic limit. While this proposed explanation for the identified upper rate limit on cellular Gibbs energy dissipation rate awaits experimental validation, it offers an intriguing perspective of how metabolic activity can globally affect biomolecular functions and will hopefully spark new research.     

嗜碱微生物多样性及其应用

碱性环境最适生长的微生物称为嗜碱微生物,这些微生物主要分布在天然碱湖、碱性沙漠和土壤中。除细菌外已分离到各种各样的微生物,包括藻类、放线菌、真菌、酵母和病毒。为了适应碱性因子它们具有特殊的细胞结构和生理机能。因此,研究适应机理并利用其特殊机能具有重要的理论和实际意义,嗜碱微生物能产生多种碱性酶和其他生物活性物质,有着广阔的应用前景。     

Broad‐substrate screen as a tool to identify substrates for bacterial Gcn5‐related N‐acetyltransferases with unknown substrate specificity

Due to a combination of efforts from individual laboratories and structural genomics centers, there has been a surge in the number of members of the Gcn5‐related acetyltransferasesuperfamily that have been structurally determined within the past decade. Although the number of three‐dimensional structures is increasing steadily, we know little about the individual functions of these enzymes. Part of the difficulty in assigning functions for members of this superfamily is the lack of information regarding how substrates bind to the active site of the protein. The majority of the structures do not show ligand bound in the active site, and since the substrate‐binding domain is not strictly conserved, it is difficult to predict the function based on structure alone. Additionally, the enzymes are capable of acetylating a wide variety of metabolites and many may exhibit promiscuity regarding their ability to acetylate multiple classes of substrates, possibly having multiple functions for the same enzyme. Herein, we present an approach to identify potential substrates for previously uncharacterized members of the Gcn5‐related acetyltransferase superfamily using a variety of metabolites including polyamines, amino acids, antibiotics, peptides, vitamins, catecholamines, and other metabolites. We have identified potential substrates for eight bacterial enzymes of this superfamily. This information will be used to further structurally and functionally characterize them.