Efficient,environmentally-friendly and specific valorization of lignin: promising role of non-radical lignolytic enzymes

Lignin is the second most abundant bio-resource in nature. It is increasingly important to convert lignin into high value-added chemicals to accelerate the development of the lignocellulose biorefinery. Over the past several decades, physical and chemical methods have been widely explored to degrade lignin and convert it into valuable chemicals. Unfortunately, these developments have lagged because of several difficulties, of which high energy consumption and non-specific cleavage of chemical bonds in lignin remain the greatest challenges. A large number of enzymes have been discovered for lignin degradation and these are classified as radical lignolytic enzymes and non-radical lignolytic enzymes. Radical lignolytic enzymes, including laccases, lignin peroxidases, manganese peroxidases and versatile peroxidases, are radical-based bio-catalysts, which degrade lignins through non-specific cleavage of chemical bonds but can also catalyze the radical-based re-polymerization of lignin fragments. In contrast, non-radical lignolytic enzymes selectively cleave chemical bonds in lignin and lignin model compounds and, thus, show promise for use in the preparation of high value-added chemicals. In this mini-review, recent developments on non-radical lignolytic enzymes are discussed. These include recently discovered non-radical lignolytic enzymes, their metabolic pathways for lignin conversion, their recent application in the lignin biorefinery, and the combination of bio-catalysts with physical/chemical methods for industrial development of the lignin refinery.     

Activation of procarcinogens by human cytochrome P450 enzymes

Enzymatic transformation of most chemical carcinogens is requisite to the formation of electrophiles that cause genotoxicity, and the cytochrome P450 (P450) enzymes are the most prominent enzymes involved in such activation reactions. During the past 15 years the human P450 enzymes have been extensively characterized. Considerable evidence exists that the variation in activity of these enzymes can have important consequences in the actions of drugs. Other studies have been concerned with the activation of procarcinogens by human P450s. Assignments of roles of particular P450s in the metabolism of chemical carcinogens are discussed, along with the current state of evidence for relationships of particular P450s with human cancer.     

Total chemical synthesis,refolding, and crystallographic structure of fully active immunophilin calstabin 2 (FKBP12.6)

Synthetic biology (or chemical biology) is a growing field to which the chemical synthesis of proteins, particularly enzymes, makes a fundamental contribution. However, the chemical synthesis of catalytically active proteins (enzymes) remains poorly documented because it is difficult to obtain enough material for biochemical experiments. We chose calstabin, a 107‐amino‐acid proline isomerase, as a model. We synthesized the enzyme using the native chemical ligation approach and obtained several tens of milligrams. The polypeptide was refolded properly, and we characterized its biophysical properties, measured its catalytic activity, and then crystallized it in order to obtain its tridimensional structure after X‐ray diffraction. The refolded enzyme was compared to the recombinant, wild‐type enzyme. In addition, as a first step of validating the whole process, we incorporated exotic amino acids into the N‐terminus. Surprisingly, none of the changes altered the catalytic activities of the corresponding mutants. Using this body of techniques, avenues are now open to further obtain enzymes modified with exotic amino acids in a way that is only barely accessible by molecular biology, obtaining detailed information on the structure‐function relationship of enzymes reachable by complete chemical synthesis.     

Origins of specificity and promiscuity in metabolic networks

How enzymes have evolved to their present form is linked to the question of how pathways emerged and evolved into extant metabolic networks. To investigate this mechanism, we have explored the chemical diversity present in a largely unbiased data set of catalytic reactions processed by modern enzymes across the tree of life. In order to get a quantitative estimate of enzyme chemical diversity, we measure enzyme multispecificity or promiscuity using the reaction molecular signatures. Our main finding is that reactions that are catalyzed by a highly specific enzyme are shared by poorly divergent species, suggesting a later emergence of this function during evolution. In contrast, reactions that are catalyzed by highly promiscuous enzymes are more likely to appear uniformly distributed across species in the tree of life. From a functional point of view, promiscuous enzymes are mainly involved in amino acid and lipid metabolisms, which might be associated with the earliest form of biochemical reactions. In this way, results presented in this paper might assist us with the identification of primeval promiscuous catalytic functions contributing to life's minimal metabolism.     

Enzyme-catalyzed detoxication reactions: mechanisms and stereochemistry

Enzyme catalyzed detoxication reactions are one of the primary defenses organisms have against chemical insult. This article reviews current chemical approaches to understanding the cooperative role of enzymes in the metabolism of foreign compounds. Emphasis is placed on chemical and stereochemical studies which help elucidate the mechanism of action and active-site topologies of the detoxication enzymes. The stereoselectivity of the cytochromes P-450 and flavin containing monooxygenases as well as the role of hemoglobin and lipid peroxidation in the primary metabolism of xenobiotics is discussed. Current knowledge of the mechanism and stereoselectivity of epoxide hydrolase is also presented. Three enzymes involved in secondary metabolism of xenobiotics, UDP-glucuronosyltransferase, sulfotransferase and glutathione S-transferase are discussed with particular emphasis on active site topology and cooperative participation with the enzymes of primary metabolism.     

Design of glycolysis

The design of the glycolytic pathway resulting from the continuous refinement of evolution is discussed with regard to three aspects. 1. Functional and structural properties of individual enzymes. The catalytic constants of the glycolytic enzymes are remarkably optimized; the turnover numbers are within one order of magnitude. The same is true for the molarities of catalytic centres in the cytosol, as is noted for yeast. Functional properties of the enzymes are reflected in their tertiary and quaternary structures. 2. Regulatory mechanisms of single enzymes. A classification of the various types of enzymic control mechanisms operating in the glycolytic pathway is given. In addition to the usual Michaelis-Menten saturation kinetics and the various types of inhibition there is control by positive and negative effectors based on oligomeric structures (fast acting, fine control) as well as regulation by chemical interconversion structures (fast acting, fine control) as well as regulation by chemical based on enzymes cascades (slow acting, very effective). 3. Functional and regulatory mechanisms of the whole glycolytic reaction pathway. A prominent feature is the high enzyme:substrate ratio, which guarantees fast response times. However, a quantitative treatment of the overall kinetics is limited by an incomplete knowledge of the enzymes' dynamic and chemical compartmentation as well as some of their control properties. From an analysis of the oscillatory state, certain control points in the glycolytic chain can be located that coincide with major branching points to other metabolic pathways. These points are controlled by fast-acting cooperative enzymes that operate in a flip-flop mechanism together with the respective antagonistic enzymes, preventing futile cycles. The gating enzymes leading to the glycogen store and the citric acid cycle are of the slow-acting but very effective interconvertible type. The combination of all the complex and intricate features of design yields a glycolytic network that enables the cell to respond to its various metabolic needs quickly, effectively and economically.     

Activity-based protein profiling: an enabling technology in chemical biology research

Activity-based protein profiling (ABPP) is one of the main driving forces in chemical biology and one of the most visible areas where organic chemistry contributes to chemical biology research. In recent years, ABPP research has gradually made the transfer from the relatively easy target enzymes (for instance serine hydrolases, cysteine and threonine proteases) toward targeting enzymes that are intrinsically more difficult to address. These include less abundant enzymes, enzymes that do not employ a nucleophilic amino acid residue in their active site and enzymes more particular with respect to their substrate. At the same time, ABPP has started to make a tangible impact on clinical research.     

Novel biocatalysis by database mining

Broad-based adoption of biocatalytic methods will require widely available database tools, analogous to previous efforts compiling information for the facilitation of chemical synthesis. The analog to chemical reagents are enzymes. The analog to chemical synthetic routes are metabolic pathways. The free on-line database BRENDA exemplifies efforts to compile relevant information on enzymes for biocatalytic purposes. Likewise, the University of Minnesota Biocatalysis/Biodegradation Database focuses on novel enzymes and metabolic pathways useful in environmental and industrial biotechnology. The development of biocatalytic protocols will be facilitated by the increasing availability of well-curated database information on enzymatic enantioselectivity and capabilities for transforming disparate chemical functional groups.     

Searching for amino acid sequence motifs among enzymes: the Enzyme-Reaction Database

Recently we have constructed a database—the Enzyme–ReactionDatabase–which links a chemical structure to amino acidsequences of enzymes that recognize the chemical structure astheir ligand. The total number of enzymes registered in thedatabase is 1103 with 6668 NBRF–PIR entry codes and 1756chemical compounds. The chemical structures and chemical namesfor 842 compounds are registered in the Chemical–StructureDatabase on the MACCS system. For each enzyme, the sequenceswere divided into clusters, and multiply aligned in each clusterto extract a conserved sequence. A total of 158 781 five–residue–longfragments were constructed from 433 conserved sequences andcompared among different clusters of different enzymes. Oneof these motifs shared by different enzymes S–G–G–L–D.The motif was conserved in both argininosuccinate synthase (EC6.3.4.5 [EC] ) and asparagine synthase (glutamine–hydrolysing)(EC 6.3.5.4 [EC] ). This result showed that the database was usefulfor the analysis of the relationship between chemical structuresand amino acid sequence motifs.     

Chemoenzymatic methods for site-specific protein modification

In the past decade, numerous chemical technologies have been developed to allow the site-specific post-translational modification of proteins. Traditionally covalent chemical protein modification has been accomplished by the attachment of synthetic groups to nucleophilic amino acids on protein surfaces. These chemistries, however, are rarely sufficiently selective to distinguish one residue within a literal sea of chemical functionality. One solution to this problem is to introduce a unique chemical handle into the target protein that is orthogonal to the remainder of the proteome. In practice, this handle can be a novel peptide sequence, which forms a 'tag' that is selectively and irreversibly modified by enzymes. Furthermore, if the enzymes can tolerate substrate analogs, it becomes possible to engineer chemically modified proteins in a site-specific fashion. This review details the significant progress in creating techniques for the chemoenzymatic generation of protein-small molecule constructs and provides examples of novel applications of these methodologies.     

Effects of chemical modification on enzymatic activities and stabilities

The influence of chemical modification on the initial specific activity, residual activity, and deactivation kinetics of various enzymes is analyzed using a series mechanism. This straightforward multistate sequential model presented is consistent with the enzyme deactivation data obtained from different fields. The enzymes are placed in five different categories depending on the effect of chemical modification on initial specific activity and residual activity or stability. Wherever possible, structure-function relationships are described for the enzymes in the different categories. The categorization provides one avenue that leads to further physical insights into enzyme deactivation processes and into the enzyme structure itself.     

Biotransformations of rifamycins: process possibilities

Rifampicin, an important antibiotic, is manufactured by chemical conversion of rifamycin S which is obtained by the chemical modification of rifamycin B. Rifamycin B is a product of Nocardia mediterranei fermentations. The chemical conversion of rifamycin B to rifamycin S has many disadvantages: Strong acidic conditions are required, heavy foam formation accompanies transformation and the yields are low. This review highlights the developments in alternative, biochemical transformations using enzymes and cells; the main focus is on transformations carried out by rifamycin oxidase.     

On the relationship between thermal stability and catalytic power of enzymes

The possible relationship between the thermal stability and the catalytic power of enzymes is of great current interest. In particular, it has been suggested that thermophilic or hyperthermophilic (Tm) enzymes have lower catalytic power at a given temperature than the corresponding mesophilic (Ms) enzymes, because the thermophilic enzymes are less flexible (assuming that flexibility and catalysis are directly correlated). These suggestions presume that the reduced dynamics of the thermophilic enzymes is the reason for their reduced catalytic power. The present paper takes the specific case of dihydrofolate reductase (DHFR) and explores the validity of the above argument by simulation approaches. It is found that the Tm enzymes have restricted motions in the direction of the folding coordinate, but this is not relevant to the chemical process, since the motions along the reaction coordinate are perpendicular to the folding motions. Moreover, it is shown that the rate of the chemical reaction is determined by the activation barrier and the corresponding reorganization energy, rather than by dynamics or flexibility in the ground state. In fact, as far as flexibility is concerned, we conclude that the displacement along the reaction coordinate is larger in the Tm enzyme than in the Ms enzyme and that the general trend in enzyme catalysis is that the best catalyst involves less motion during the reaction than the less optimal catalyst. The relationship between thermal stability and catalysis appears to reflect the fact that to obtain small electrostatic reorganization energy it is necessary to invest some folding energy in the overall preorganization process. Thus, the optimized catalysts are less stable. This trend is clearly observed in the DHFR case.     

Chemical and genomic evolution of enzyme-catalyzed reaction networks

There is a tendency that a unit of enzyme genes in an operon-like structure in the prokaryotic genome encodes enzymes that catalyze a series of consecutive reactions in a metabolic pathway. Our recent analysis shows that this and other genomic units correspond to chemical units reflecting chemical logic of organic reactions. From all known metabolic pathways in the KEGG database we identified chemical units, called reaction modules, as the conserved sequences of chemical structure transformation patterns of small molecules. The extracted patterns suggest co-evolution of genomic units and chemical units. While the core of the metabolic network may have evolved with mechanisms involving individual enzymes and reactions, its extension may have been driven by modular units of enzymes and reactions.     

应用生物法合成内酯化合物

内酯是广泛存在于自然界中具有生物活性的一类化合物。由于大多数内酯化合物具有手性,用化学方法合成不仅过程复杂,而且产率也不高。利用酶反应的特异性,应用生物法合成内酯化合物具有很好的应用前景,其中包括微生物次生代谢合成内酯,脂肪酸生物转化合成内酯和脂肪酶在有机相中催化羟基脂肪酸形成内酯。本文报道这些领域的进展。     

Enzyme immobilization for biodiesel production

Biodiesel has attracted more and more attention in recent years because of its biodegradability, environmentally friendliness, and renewability. Contrary to the conventional chemical catalysis method to produce biodiesel, the biochemical catalysis method developed quickly in the past decade and many immobilized enzymes are commercially available to meet the large-scale industrialization of biodiesel. This review is focusing on the current status of biodiesel production by biochemical catalysis method, especially the commercial enzyme and its immobilization for biodiesel production. Consequently, we believe that biochemical catalysis with immobilized enzymes is bound to be an alternative method instead of chemical catalysis in biodiesel production in the near future.     

嵌合体纤溶酶的研究进展

嵌合体纤溶酶是采用基因重组技术或化学偶联的方法将纤溶酶与其它的功能多肽(舍有酶原结构域,具有抗凝血酶、抗血小板聚集活性,能特异识别纤维蛋白的多肽)结合起来所形成的嵌合体蛋白质．它尽可能地保留了分子中每一组分的生物活性,增强了纤溶酶的溶栓功效,较大程度地克服了临床溶栓药物在某些方面的不足,是目前溶栓药物研究领域中的热点。     

Directed Evolution of an Enantioselective Bacillus subtilis Lipase

Chiral compounds are of steadily increasing importance to the chemical industry, in particular for the production of pharmaceuticals. Where do these compounds come from? Apart from natural resources, two synthetic strategies are available: asymmetric chemical catalysis using transition metal catalysts and biocatalysis using enzymes. In the latter case, screening programs have identified a number of enzymes. However, their enantioselectivity is often not high enough for a desired reaction. This problem can be solved by applying directed evolution to create enantioselective enzymes as shown here for a lipase from Bacillus subtilis. The reaction studied was the asymmetric hydrolysis of meso-1,4-diacetoxy-Zcyclopentene with the formation of chiral alcohols which were detected by electrospray ionization mass spectrometry. Iterative cycles of random mutagenesis and screening allowed the identification of several variants with improved enantioselectivities. In parallel, we have started to use X-ray structural data to simulate the Bacillus subtilis lipase A-catalyzed substrate hydrolysis by using quantum mechanical and molecular mechanical calculations. This combined approach should finally enable us to devise more efficient strategies for the directed evolution of enantioselective enzymes.     

Engineering enzymes

Could we design and construct enzymes to catalyse any desired reaction? Compared with organic chemical catalysts, enzymes are highly specific and work in dilute aqueous solutions at ambient temperatures. Substrates are brought together from solution to precise orientations in the active site of an enzyme and the amino acid side-chains of the enzyme may assist catalysis by attacking or destabilizing substrate bonds. In principle, a novel enzyme could be constructed de novo or from pre-existing enzymes. Altering enzymes by recombinant DNA techniques offers most chance of success.     

Cofactor binding modulates the conformational stabilities and unfolding patterns of NAD(+)-dependent DNA ligases from Escherichia coli and Thermus scotoductus

DNA ligases are important enzymes required for cellular processes such as DNA replication, recombination, and repair. NAD(+)-dependent DNA ligases are essentially restricted to eubacteria, thus constituting an attractive target in the development of novel antibiotics. Although such a project might involve the systematic testing of a vast number of chemical compounds, it can essentially gain from the preliminary deciphering of the conformational stability and structural perturbations associated with the formation of the catalytically active adenylated enzyme. We have, therefore, investigated the adenylation-induced conformational changes in the mesophilic Escherichia coli and thermophilic Thermus scotoductus NAD(+)-DNA ligases, and the resistance of these enzymes to thermal and chemical (guanidine hydrochloride) denaturation. Our results clearly demonstrate that anchoring of the cofactor induces a conformational rearrangement within the active site of both mesophilic and thermophilic enzymes accompanied by their partial compaction. Furthermore, the adenylation of enzymes increases their resistance to thermal and chemical denaturation, establishing a thermodynamic link between cofactor binding and conformational stability enhancement. Finally, guanidine hydrochloride-induced unfolding of NAD(+)-dependent DNA ligases is shown to be a complex process that involves accumulation of at least two equilibrium intermediates, the molten globule and its precursor.