Enzymatic hydroxylation reactions

During the past 18 months, considerable progress has been made in the understanding of the key enzyme-substrate interactions that control the regioselectivity and stereoselectivity of the hydroxylation reaction performed by cytochrome-P450-dependent enzymes of mammalian origin. The manipulation of microbial hydroxylating enzymes, in both whole-cell and cell-free environments, has also been examined in the context of controlling the regioselectivity and stereoselectivity of the hydroxylation reaction. Several new applications for hydroxylating enzymes have been reported, and the construction of chimeric hydroxylating enzymes has been used both for mechanistic studies and for the production of enzymes with high hydroxylating activity for a defined substrate.     

Plant aspartic proteinases: enzymes on the way to a function

Plant aspartic proteinases have been characterized from seeds, flowers and leaves of a number of different species. The enzymes are generally either monomeric or heterodimeric, containing two peptides processed from the same precursor protein. The plant enzymes, like their mammalian and microbial counterparts, are active at acidic pH and inhibited by a class specific inhibitor pepstatin A. Plant aspartic proteinases are generally either secreted or targeted to the vacuolar/protein storage body compartment. The primary sequences of many of these enzymes have been determined and are very homologous with each other as well as with enzymes from mammalian and microbial origins. Plant aspartic proteinases, however, have a very unique plant specific region, which is not found in mammalian, microbial, or viral aspartic proteinases. The function of this region has not been elucidated. A role for these plant enzymes in protein processing or degradation has been proposed, however, more studies are required to confirm their in vivo functions. Recent intriguing results suggest possible roles for these enzymes in programmed cell-death of tissues and in pathogen resistance.     

Enzymes in the synthesis of bioactive compounds: the prodigious decades

The growing demand for enantiomerically pure pharmaceuticals has impelled research on enzymes as catalysts for asymmetric synthetic transformations. However, the use of enzymes for this purpose was rather limited until the discovery that enzymes can work in organic solvents. Since the advent of the PCR the number of available enzymes has been growing rapidly and the tailor-made biocatalysts are becoming a reality. Thus, it has been possible the use of enzymes for the synthesis of new innovative medicines such as carbohydrates and their incorporation to modern methods for drug development, such as combinatorial chemistry. Finally, the genomic research is allowing the manipulation of whole genomes opening the door to the combinatorial biosynthesis of compounds. In this review, our intention is to highlight the main landmarks that have led to transfer the chemical efficiency shown by the enzymes in the cell to the synthesis of bioactive molecules in the lab during the last 20 years.     

Structure/function studies on enzymes in the diaminopimelate pathway of bacterial cell wall biosynthesis.

Within the past 18 months work has continued on the structure and mechanisms of enzymes involved in the diaminopimelic acid/lysine biosynthetic pathway. A novel structure has been determined for a PLP-independent epimerase, and structures with bound substrates have been solved for two other enzymes. Additionally, new studies have appeared describing the chemical mechanisms of three enzymes in the pathway.     

Sulfotransferases, sulfatases and formylglycine-generating enzymes: a sulfation fascination

Sulfotransferases and sulfatases are the major enzymes responsible for sulfate transfer processes. The past two years have seen the elucidation of new functions for these enzymes, and a great progression in their structural characterization, which confirms that these two types of enzymes possess a highly conserved fold. For catalytic activity, sulfatases must contain a formylglycine residue, which is generated by various formylglycine-generating enzymes. Mechanistic and structural details have recently been obtained for a group of cofactor-independent formylglycine-generating enzymes termed FGEs. Finally, an increasing light has been cast upon the mechanism of sulfatase inactivation by a group of clinically important agents, the aryl sulfamates.     

The glutathione system. I. Synthesis, transport, glutathione transferases, glutathione peroxidases

During the last 10–15 years significant progress has been achieved in all directions of studies of the glutathione system. A series of new enzymes involved into metabolism of glutathione has been discovered. Many of these enzymes are polyfunctional and their new activities have been recognized. The enzymes interact with hormones and signal transduction systems. Significant progress has been achieved in the studies of intracellular, intercellular and inter-organ transport. The important achievement is employment of not only selective compounds-analyzers but also gene engineering methods for identification of new functions.     

DNA-dependent RNA polymerases isolated from yeast mitochondria

Purified preparations of yeast mitochondria yield three species of DNA-dependent RNA polymerases. These enzymes have been separated and purified to homogeneity for analysis of their properties and for comparison with the properties of nuclear preparations of yeast RNA polymerases. Three enzymes have been separated by DEAE-Sephadex chromatography of each fraction. Both nuclear and mitochondrial preparations yield three components with nearly identical elution properties. The distributions of enzyme activity on DEAE-Sephadex chromatography differ with the three nuclear peaks, being found in ratios (uncorrected for the effect of increasing salt concentration) of 8:85:7 and the mitochondrial peaks in ratios of 8:32:60 at late log phase of growth under optimized conditions in which protease inhibitors and an antioxidant were included. The type of mitochondrial enzymes in 3-day-old cells differed from those grown to late logarithmic phase. It has been established that the enzymes of the mitochondrial preparation are associated with the membrane fraction. While extraction with 0.5 m KCl solubilizes considerable enzyme activity, greatly enhanced yields of enzyme MIII are obtained by addition of the antioxidant 2,6-di-t-butyl-4-hydroxymethyl phenol during enzyme extraction. Inhibition of protease activity has also been shown to have a major effect on the yield and distribution of enzymes obtained from mitochondrial preparations. The mitochondrial preparations of yeast polymerases are generally similar but not identical to corresponding nuclear polymerases in subunit molecular weights, inhibitor sensitivities, and in DNA template dependence. Comparative studies of nuclear and mitochondrial polymerases clearly establish that differences do exist among the isolated enzymes of these classes. It has not been ruled out to date that these enzymes may be derived in part or in total from the same cytoplasmic subunit pool, nor has it been established that any of these enzymes function in mitochondria in vivo.     

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.     

保幼激素生物合成研究进展

保幼激素（juvenile hormone,JH）是存在于昆虫、甲壳动物和部分植物体内的倍半萜类衍生物。在昆虫和甲壳动物体内,保幼激素主要调节变态和生殖活动。在植物体内,则可能作为异株克生物质发挥作用。保幼激素主要通过细胞质内的甲羟戊酸途径（MVA）合成,植物质体内存在萜类合成的1-去氧木糖-5-磷酸途径（DXP）。MVA和DXP途径通过单向质子协同运输系统进行协调,使DXP途径中形成的前体化合物参与MVA途径的倍半萜合成。JH生物合成的主要步骤己基本查明,但与合成相关的酶学研究还较薄弱。生物合成酶的分子生物学是近来研究的热点,相关酶的cDNA克隆已有报道。JH生物合成酶的进一步研究有助于查明JH生物合成调控机制,深化对节肢动物生殖的理解,还可为新型杀虫剂开发提供可能的靶标。     

Haloperoxidases and their role in biotransformation reactions

The past year has seen further structural characterisation of both nonmetal and vanadium haloperoxidase enzymes to add to that already known for the haem- and vanadium-containing enzymes. Exploitation of these enzymes for halogenation, sulfoxidation, epoxidation, oxidation of indoles and other biotransformations has increased as more information on their catalytic mechanism has been obtained.     

The molecular architecture of major enzymes from ajmaline biosynthetic pathway

The biosynthetic pathway leading to the monoterpenoid indole alkaloid ajmaline in Rauvolfia serpentiin serpentina is one of the most studied in the field of natural product biosynthesis. Ajmaline has a complex structure which is based on a six-membered ring system harbouring nine chiral carbon atoms. There are about fifteen enzymes involved, including some involving the side reactions of the ajmaline biosynthetic pathway. All enzymes exhibit pronounced substrate specificity. In the recent years isolation and sequencing of their cDNAs has allowed a detailed sequence analysis and comparison with functionally related and occasionally un-related enzymes. Site-directed mutations of several of the ajmaline-synthesizing enzymes have been performed and their catalytic residues have been identified. Success with over-expression of the enzymes was an important step for their crystallization and structural analysis by X-ray crystallography. Crystals with sufficient resolution were obtained from the major enzymes of the pathway. Strictosidine synthase has a 3D-structure with a six-bladed β-propeller fold the first time such a fold found in the plant kingdom. Its ligand complexes with tryptamine and secologanin, as well as structure-based sequence alignment, indicate a possible evolutionary relationship to several primary sequence-unrelated structures with this fold. The structure of strictosidine glucosidase was determined and its structure has as a (β/α)₈ barrel fold. Vinorine synthase provides the first 3D structure of a member of BAHD enzyme super-family. Raucaffricine glucosidase involved in a side-route of ajmaline biosynthesis has been crystallized. The ajmaline biosynthetic pathway is an outstanding example where many enzymes 3D-structure have been known and where there is a real potential for protein engineering to yield new alkaloid.     

S-adenosylmethionine radical enzymes

The role of S-adenosylmethionine (SAM) as a precursor to organic radicals, generated by one-electron reduction of SAM and subsequent fission to form 5'-deoxyadenosyl radical and methionine, has been known for some time. Only recently, however, has it become apparent how widespread such enzymes are, and what a wide range of chemical reactions they catalyze. In the last few years several new SAM radical enzymes have been identified. Spectroscopic and kinetic investigations have begun to uncover the mechanism by which an iron sulfur cluster unique to these enzymes reduces SAM to generate adenosyl radical. Most recently, the first X-ray structures of SAM radical enzymes, coproporphyrinogen-III oxidase, and biotin synthase have been solved, providing a structural framework within which to interpret mechanistic studies.     

Application of enzymes in the pulp and paper industry

The pulp and paper industry processes huge quantities of lignocellulosic biomass every year. The technology for pulp manufacture is highly diverse, and numerous opportunities exist for the application of microbial enzymes. Historically, enzymes have found some uses in the paper industry, but these have been mainly confined to areas such as modifications of raw starch. However, a wide range of applications in the pulp and paper industry have now been identified. The use of enzymes in the pulp and paper industry has grown rapidly since the mid 1980s. While many applications of enzymes in the pulp and paper industry are still in the research and development stage, several applications have found their way into the mills in an unprecedented short period of time. Currently the most important application of enzymes is in the prebleaching of kraft pulp. Xylanase enzymes have been found to be most effective for that purpose. Xylanase prebleaching technology is now in use at several mills worldwide. This technology has been successfully transferred to full industrial scale in just a few years. The enzymatic pitch control method using lipase was put into practice in a large-scale paper-making process as a routine operation in the early 1990s and was the first case in the world in which an enzyme was successfully applied in the actual paper-making process. Improvement of pulp drainage with enzymes is practiced routinely at mill scale. Enzymatic deinking has also been successfully applied during mill trials and can be expected to expand in application as increasing amounts of newsprint must be deinked and recycled. The University of Georgia has recently opened a pilot plant for deinking of recycled paper. Pulp bleaching with a laccase mediator system has reached pilot plant stage and is expected to be commercialized soon. Enzymatic debarking, enzymatic beating, and reduction of vessel picking with enzymes are still in the R&D stage but hold great promise for reducing energy. Other enzymatic applications, i.e., removal of shives and slime, retting of flax fibers, and selective removal of xylan, are also expected to have a profound impact on the future technology of the pulp and paper-making process.     

The use of step enzymes as markers during meiosis and ascospore formation in Saccharomyces cerevisiae.

The activities of ornithine aminotransferase, sucrase and acid and alkaline phosphatases have been studied throughout sporulation in Saccharomyces cerevisiae. The same enzymes were monitored during synchronous vegetative growth. Each of these enzymes has been demonstrated to increase in a 'step' manner during both growth and sporulation. Alkaline phosphatase increased in a two-step manner whereas the others increased in a single step. The times of increase of these enzymes formed a similar sequence during both sporulation and growth. It has been proposed that these enzymes are under a common mechanism of control during growth and sporulation and that the sequence of enzyme appearance may be used as markers of the sporulation process.     

Biochemical and genetical approaches to the mechanism of action of penicillin

Since the discovery in 1965 that penicillin inhibits the transpeptidation reaction in peptidoglycan synthesis, a considerable effort has been put into the purification of enzymes that catalyse this reaction. This has resulted in the recognition that bacteria possess multiple forms of these penicillin-sensitive enzymes and has made it difficult to identify the precise target that penicillin inactivates to kill the organism. Recently penicillin-sensitive enzymes have been detected and studies as penicillin-binding proteins on sodium dodecyl sulphate polyacrylamide gels. The availability of this convenient method for identifying penicillin-sensitive enzymes has allowed biochemical and genetical approaches to be used to dissect their roles in the lethal effects of penicillin and other beta-lactam antibiotics. Three penicillin-binding proteins (1 B, 2 and 3) have been identified as killing targets for penicillin in Escherichia coli, whereas four other binding proteins are not implicated in the mechanism of action of the antibiotic. The complex biological effects that beta-lactam antibiotics produce on the growth of E. coli can be explained by their interaction with the three killing targets. Progress in the correlation of penicillin-binding proteins with penicillin-sensitive enzymes and in the development of strains of E. coli that overproduce penicillin-binding proteins is discussed.     

Plant glycosidases acting on protein-linked oligosaccharides

Glycosidases have been used as invaluable tools in glycobiology research for decades, and their role in glycoprotein maturation has been amply studied. The molecular biological coverage of this large group of enzymes has only recently reached an appreciable level. In this review, we present an overview of plant glycosidases, whose DNA/protein sequence has been identified and for which recombinant enzymes have been characterized. The physiological role in the maturation of glycoproteins is discussed as well as the biotechnological prospects arising from knowing the enzymes responsible for the removal of terminal N-acetylglucosamine residues. The current knowledge on plant fucosidases and of the first bits of information on glycosidases acting on arabinogalactan proteins is presented.     

嗜热酶的耐热机理

酶蛋白在高温下的不稳定性是影响其广泛应用的主要瓶颈,嗜热酶因为独特的性质而被作为热稳定研究的极好材料。了解嗜热酶的热稳定性机制,对于采用酶工程定向设计、改造酶具有重要的意义。嗜热酶的热稳定性并不是由单一因素决定的,氨基酸组成、氢键、离子对、二硫键等都是影响嗜热酶热稳定性的重要因素。相对于嗜温酶,嗜热酶更多地采用寡聚体的形式。     

Bacteriolytic enzymes from the blood plasma of the sheep

Bacteriolytic factors from the blood plasma of healthy sheep have been studied. Three enzymes not described earlier in the literature have been identified and characterized. Two enzymes exhibit activity toward Escherichia coli and have molecular weights of 15 ± 2 kDa. The third enzyme that exhibits activity toward E. coli and Micrococcus luteus has a molecular weight of 34 ± 4 kDa. The kinetic parameters of bacterial lysis for all enzymes have been determined; in particular, optimal pH values for each of the substrates used have been found. For the identification of the enzymes, trypsinolysis and a mass-spectroscopic study of fragments have been carried out. The results were compared with the data on sheep proteins available in the Swiss-Prot, NCBI, and MSDB databases.     

The kinetic model of the shikimate pathway as a tool to optimize enzyme assays for high-throughput screening

Four-enzyme section of the shikimate pathway (Aro B, D, E, and K) of Streptococcus pneumoniae has been studied. Kinetic properties of the individual enzymes and three- and four-enzyme linked reactions have been characterized in vitro. On the basis of the data measured in spectrophotometric and LC-MS experiments, kinetic mechanisms of the enzymes have been suggested and all kinetic parameters have been identified. Kinetic models for these three- and four-enzyme sections of the shikimate pathway have been constructed and validated. The model of the four-enzyme section of shikimate pathway has been employed to design an inhibition-sensitive reconstituted pathway for a high-throughput screening effort on the shikimate pathway. It was demonstrated that using the model it was possible to optimize this reconstituted pathway in such a way to provide equal sensitivity of the enzymes to inhibition.