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.     

Enzymatic halogenation catalyzed via a catalytic triad and by oxidoreductases

During the search for haloperoxidases in bacteria we detected a type of enzymes that catalyzed the peroxide-dependent halogenation of organic substrates. However, in contrast to already known haloperoxidases, these enzymes do not contain a prosthetic group or metal ions nor any other cofactor. Biochemical and molecular genetic studies revealed that they contain a catalytic triad consisting of a serine, a histidine, and an aspartate. The reaction they catalyze is actually the perhydrolysis of an acetic acid serine ester leading to the formation of peracetic acid. As a strong oxidizing agent the enzymatically formed peracetic acid can oxidize halide ions, resulting in the formation of hypohalous acid which then acts as the actual halogenating agent. Since hypohalous acid is also formed by the heme- and vanadium-containing haloperoxidases, enzymatic halogenation catalyzed by haloperoxidases and perhydrolases in general lacks substrate specificity and regioselectivity. However, detailed studies on the biosynthesis of several halometabolites led to the detection of a novel type of halogenases. These enzymes consist of a two-component system and require NADH and FAD for activity. Whereas the gene for one of the components is part of the biosynthetic cluster of the halometabolite, the second component is an enzyme which is also present in bacteria from which no halometabolites have ever been isolated, like Escherichia coli. In contrast to haloperoxidases and perhydrolases the newly detected NADH/FAD-dependent halogenases are substrate-specific and regioselective and might provide ideal tools for specific halogenation reactions.     

Molecular mechanisms of enzyme-catalysed halogenation

Since their discovery, halogenated metabolites have been somewhat of a biological peculiarity and it is only now that we are beginning to realize the full extent of their medicinal value. With the exception of the well characterized haloperoxidases, most of the biosynthetic enzymes and mechanisms responsible for the halogenations have remained elusive. The crystal structures of two functionally diverse halogenases have been recently solved, providing us with new and exciting mechanistic detail. This new insight has the potential to be used both in the development of biomimetic halogenation catalysts and in engineering halogenases, and related enzymes, to halogenate new substrates. Interestingly, these new structures also illustrate how the evolution of these enzymes mirrors that of the monooxygenases, where the cofactor is selected for its ability to generate a powerful oxygenating species. In this highlight article we will examine the proposed catalytic mechanisms of the halogenases and how these relate to their structures. In addition, we will consider how this chemistry might be harnessed and developed to produce novel enzymatic activity.     

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.     

A Fivefold Parallelized Biosynthetic Process Secures Chlorination of Armillaria mellea (Honey Mushroom) Toxins

The basidiomycetous tree pathogen Armillaria mellea (honey mushroom) produces a large variety of structurally related antibiotically active and phytotoxic natural products, referred to as the melleolides. During their biosynthesis, some members of the melleolide family of compounds undergo monochlorination of the aromatic moiety, whose biochemical and genetic basis was not known previously. This first study on basidiomycete halogenases presents the biochemical in vitro characterization of five flavin-dependent A. mellea enzymes (ArmH1 to ArmH5) that were heterologously produced in Escherichia coli. We demonstrate that all five enzymes transfer a single chlorine atom to the melleolide backbone. A 5-fold, secured biosynthetic step during natural product assembly is unprecedented. Typically, flavin-dependent halogenases are categorized into enzymes acting on free compounds as opposed to those requiring a carrier-protein-bound acceptor substrate. The enzymes characterized in this study clearly turned over free substrates. Phylogenetic clades of halogenases suggest that all fungal enzymes share an ancestor and reflect a clear divergence between ascomycetes and basidiomycetes.     

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.     

Optimisation of halogenase enzyme activity by application of a genetic algorithm

A genetic algorithm (GA) was applied for the optimisation of an enzyme assay composition respectively the enzyme activity of a recombinantly produced FADH(2)-dependent halogenating enzyme. The examined enzyme belongs to the class of halogenases and is capable to halogenate tryptophan regioselective in position 5. Therefore, the expressed trp-5-halogenase can be an interesting tool in the manufacturing of serotonin precursors. The application of stochastic search strategies (e.g. GAs) is well suited for fast determination of the global optimum in multidimensional search spaces, where statistical approaches or even the popular classical one-factor-at-a-time method often failures by misleading to local optima. The concentrations of six different medium components were optimised and the maximum yield of the halogenated tryptophan could be increased from 3.5 up to 65%.     

Lignin-enzyme interaction: Mechanism,mitigation approach,modeling, and research prospects

The adverse environmental impacts of the fossil fuel and the concerns of energy security necessitate the development of alternative clean energy sources from renewable feedstocks. Lignocellulosic biomass is a 2nd generation feedstock used in the production of biofuels and bio-based products that are conventionally derived from fossil resources. The biochemical conversion, which entails biomass pretreatment, enzymatic hydrolysis and fermentation, is one major platform used to transform lignocelluloses into biofuels. However, lignin presents many challenges to enzymatic hydrolysis leading to the need of high enzyme dose, low hydrolysis yield, low level of recyclability, high cost of enzymatic hydrolysis (because of the high cost of enzymes), and so on. Therefore, enzymatic hydrolysis, which is not cost effective, becomes one of major cost contributors. To mitigate the negative effects of lignin, extensive research has been conducted to explore the fundamental mechanisms of lignin-enzyme interactions to develop technologies to overcome the negative effects of lignin on enzymatic hydrolysis. Non-productive adsorption, which is characterized by hydrophobic, electrostatic and/or hydrogen bonding interactions, is widely known as the primary mechanism governing lignin-enzyme interactions. In addition, lignin-enzyme interaction is also influenced by steric hindrance (i.e., the physical blocking of enzyme access to carbohydrates by lignin). However, the mechanisms underlying the lignin-enzyme interactions remain unclear. This article aims to present a comprehensive review on the lignin-enzyme interactions (i.e. the mechanism, governing driving forces, modeling, and technologies for mitigating the negative effect of lignin). The current challenges inherent in this process and possible avenues of research in cellulosic biorefinery conclude this article.     

Regioselectivity of substrate hydroxylation versus halogenation by a nonheme iron(IV)–oxo complex: possibility of rearrangement pathways

Several nonheme iron enzymes and biomimetic model complexes catalyze a substrate halogenation reaction. Recent computational studies (Borowski et al. J Am Chem Soc 132:12887-12898, 2010) on α-ketoglutarate dependent halogenase proposed an initial isomerization reaction that is important to give halogenated products. We present here a series of density functional theory calculations on a biomimetic model complex-[Fe(IV)(O)(TPA)Cl](+), where TPA is tris(2-pyridylmethyl)amine-and investigate the mechanisms of substrate halogenation versus hydroxylation using the reactant and its isomer where the oxo and chloro groups have changed positions. We show here that the reactions occur on a dominant quintet spin state surface, although the reactants are in a triplet state. Despite the fact that the reactants can exist in two stable isomers with the oxo group either trans or cis to the axial ligand, they react differently with substrates, where one gives dominant hydroxylation and the other gives dominant chlorination of substrates. The ligand in the cis position of the oxo group is found to be active in the reaction mechanism and donated to the substrate during the reaction. A detailed thermochemical analysis of possible reaction mechanisms reveals that the strengths of the Fe-OH and Fe-Cl bonds in the radical intermediates are the key reasons for this regioselectivity switch of hydroxylation over halogenation. This study highlights the differences between enzymatic and biomimetic halogenases, where the former only react after an essential isomerization step, which is not necessary in model complexes.     

甲基-辅酶M还原酶结构、功能及催化机制研究进展

产甲烷古菌是目前发现唯一能产生甲烷气体的微生物,也是自然界中生物甲烷的主要贡献者。甲基-辅酶M还原酶(Methyl-coenzyme M reductase,Mcr)负责产甲烷代谢中最后一步甲烷的生成与甲烷氧化代谢中第一步甲烷的激活反应。该酶的基因高度保守,被广泛应用于古菌的鉴定与系统发育研究。其特殊的辅因子F430及催化碳氢(C-H)键裂解的酶学机制也一直备受关注。近年来,在高分辨率蛋白结构和反应过渡态结构方面的重要突破有效地推动了Mcr结构与功能的研究。特别是最新发现的激活非甲烷烷烃厌氧降解的类甲基-辅酶M还原酶(Mcr-like),引起了众多研究者对该类酶激活惰性烷烃分子机制的浓厚兴趣。因此,文中概述了Mcr结构、功能及催化机制的最新研究进展,包括新发现的Mcr-like的研究情况,并展望了Mcr/Mcr-like酶在烷烃厌氧氧化及温室气体控制方面的未来研究方向。     

Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics, and structures

The cleaning power of detergents seems to have peaked; all detergents contain similar ingredients and are based on similar detergency mechanisms. To improve detergency, modern types of heavy-duty powder detegents and automatic dishwasher detergents usually contain one or more enzymes, such as protease, amylase, cellulase, and lipase. Alkaliphilic Bacillus strains are often good sources of alkaline extracellular enzymes, the properties of which fulfil the essential requirements for enzymes to be used in detergents. We have isolated numbers of alkaliphilic Bacillus that produce such alkaline detergent enzymes, including cellulase (CMCase), protease, α-amylase, and debranching enzymes, and have succeeded in large-scale industrial production of some of these enzymes. Here, we describe the enzymatic properties, genetics, and structures of the detergent enzymes that we have developed. Received: January 22, 1998 / Accepted: February 16, 1998     

Metabolomics, genomics, proteomics, and the identification of enzymes and their substrates and products

A large proportion of the genes in any plant genome encode enzymes of primary and specialized (secondary) metabolism. Not all plant primary metabolites, those that are found in all or most species, have been identified. Moreover, only a small portion of the estimated hundreds of thousand specialized metabolites, those found only in restricted lineages, have been studied in any species. The correlative analysis of extensive metabolic profiling and gene expression profiling has proven a powerful approach for the identification of candidate genes and enzymes, particularly those in secondary metabolism. The final characterization of substrates, enzymatic activities, and products requires biochemical analysis, which has been most successful when candidate proteins have homology to other enzymes of known function. The challenges are to identify new types of enzymes and to develop biochemical techniques that are suitable for large-scale analysis.     

Aminomutases: mechanistic diversity, biotechnological applications and future perspectives

Aminomutases carry out the chemically challenging exchange of a hydrogen atom and an amine substituent present on neighboring carbon atoms. In recent years, aminomutases have been intensively investigated for their biophysical, structural and mechanistic characteristics. The reactions catalyzed by these enzymes have considerable potential for biotechnological applications. Here, we present an overview of this diverse group of enzymes, with a focus on enzymatic mechanisms and recent developments in their use in applied biocatalysis.     

人体肠道菌群来源碳水化合物活性酶（CAZYmes） 研究进展

肠道菌群是人体重要的代谢"器官",对人体的健康和疾病起着至关重要的作用.肠道菌群参与人体消化、免疫、神经系统调节机能的分子机理是特异性物质代谢通路在微生物与人体之间的协同耦合.酶是代谢通路中参与物质转化的基本功能单元,深入理解肠道菌群编码酶的分子催化机理将为以肠道菌群(或肠道酶)作为靶点的精准营养/医疗干预研究提供重要理论依据.特异性底物酶解研究表明,肠道菌群编码的酶系统不仅包含全部已知的碳水化合物活性酶(carbohydrateactive enzymes, CAZYmes)类,同时蕴含诸多潜在的新型CAZYmes.本文阐述CAZYmes的分类原则及催化机理,并主要从结构生物学方面综述人体肠道菌群来源的新型CAZYmes.     

Heme-thiolate haloperoxidases: versatile biocatalysts with biotechnological and environmental significance

Heme-thiolate haloperoxidases are undoubtedly the most versatile biocatalysts of the hemeprotein family and share catalytic properties with at least three further classes of heme-containing oxidoreductases, namely, classic plant and fungal peroxidases, cytochrome P450 monooxygenases, and catalases. For a long time, only one enzyme of this type—the chloroperoxidase (CPO) of the ascomycete Caldariomyces fumago—has been known. The enzyme is commercially available as a fine chemical and catalyzes the unspecific chlorination, bromination, and iodation (but no fluorination) of a variety of electrophilic organic substrates via hypohalous acid as actual halogenating agent. In the absence of halide, CPO resembles cytochrome P450s and epoxidizes and hydroxylates activated substrates such as organic sulfides and olefins; aromatic rings, however, are not susceptible to CPO-catalyzed oxygen-transfer. Recently, a second fungal haloperoxidase of the heme-thiolate type has been discovered in the agaric mushroom Agrocybe aegerita. The UV–Vis adsorption spectrum of the isolated enzyme shows little similarity to that of CPO but is almost identical to a resting-state P450. The Agrocybe aegerita peroxidase (AaP) has strong brominating as well as weak chlorinating and iodating activities, and catalyzes both benzylic and aromatic hydroxylations (e.g., of toluene and naphthalene). AaP and related fungal peroxidases could become promising biocatalysts in biotechnological applications because they seemingly fill the gap between CPO and P450 enzymes and act as “self-sufficient” peroxygenases. From the environmental point of view, the existence of a halogenating mushroom enzyme is interesting because it could be linked to the multitude of halogenated compounds known from these organisms.     

Enzymatic and nonenzymatic cross-linking of collagen and elastin.

Knowledge regarding the steps and mechanisms related to the intra- and interchain cross-linking of collagen and elastin has evolved steadily during the past 30 years. Recently, effort has been directed at identifying the location and types of cross-links that are found in collagen and elastin. There are two major groups of cross-links: those initiated by the enzyme lysyl oxidase and those derived from nonenzymatically glycated lysine and hydroxylysine residues. The formation of enzymatic cross-links depends on specific enzymes, amino acid sequences, and quaternary structural arrangements. The cross-links that are derived nonenzymatically occur more adventitiously and are important to pathobiological processes. Considerable progress has been made in elucidating the pathways of synthesis for several of the enzymatically mediated cross-links, as well as possible mechanisms regulating the specificity of cross-linking. Although less is known about the chemistry of cross-links arising from nonenzymatically glycated residues, recent progress has also been made in understanding possible biosynthetic pathways and control mechanisms. This review focuses on such progress and hopes to underscore the biological importance of collagen and elastin cross-linking.     

Critical view on the monochlorodimedone assay utilized to detect haloperoxidase activity

The current study aimed to identify the halogenating enzymes involved in the biosynthesis of the ambigols A, B, C and tjipanazole D, isolated from the cyanobacterium Fischerella ambigua. Haloperoxidase (HPO) activity within F. ambigua was therefore assayed spectrophotometrically by using monochlorodimedone (MCD) during protein purification. This strategy revealed the isolation of a protein positive in the MCD-assay, but an involvement in halogenating processes could not be verified. N-terminal sequencing rather demonstrated homology to cytochrome c(6) from other cyanobacteria and green algae. From our findings it thus has to be concluded that the spectrophotometrical MCD-assay routinely used to detect HPO activity may yield false positive results, mainly since the assay focuses on the decline of the educt and not on the formation of the product. Our data indicate that the reaction of MCD with proteins of the cytochrome c- family leads to unspecific products.     

Chemistry enters nucleic acids biology: Enzymatic mechanisms of RNA modification

Modified nucleotides are universally conserved in all living kingdoms and are present in almost all types of cellular RNAs, including tRNA, rRNA, sn(sno)RNA, and mRNA and in recently discovered regulatory RNAs. Altogether, over 110 chemically distinct RNA modifications have been characterized and localized in RNA by various analytical methods. However, this impressive list of known modified nucleotides is certainly incomplete, mainly due to difficulties in identification and characterization of these particular residues in low abundance cellular RNAs. In DNA, modified residues are formed by both enzymatic reactions (like DNA methylations, for example) and by spontaneous chemical reactions resulting from oxidative damage. In contrast, all modified residues characterized in cellular RNA molecules are formed by specific action of dedicated RNA-modification enzymes, which recognize their RNA substrate with high specificity. These RNA-modification enzymes display a great diversity in terms of the chemical reaction and use various low molecular weight cofactors (or co-substrates) in enzymatic catalysis. Depending on the nature of the target base and of the co-substrate, precise chemical mechanisms are used for appropriate activation of the base and the co-substrate in the enzyme active site. In this review, we give an extended summary of the enzymatic mechanisms involved in formation of different methylated nucleotides in RNA, as well as pseudouridine residues, which are almost universally conserved in all living organisms. Other interesting mechanisms include thiolation of uridine residues by ThiI and the reaction of guanine exchange catalyzed by TGT. The latter implies the reversible cleavage of the N-glycosidic bond in order to replace the initially encoded guanine by an aza-guanosine base. Despite the extensive studies of RNA modification and RNA-modification machinery during the last 20 years, our knowledge on the exact chemical steps involved in catalysis of RNA modification remains very limited. Recent discoveries of radical mechanisms involved in base methylation clearly demonstrate that numerous possibilities are used in Nature for these difficult reactions. Future studies are certainly required for better understanding of the enzymatic mechanisms of RNA modification, and this knowledge is crucial not only for basic research, but also for development of new therapeutic molecules.