Metabolic engineering of isoflavonoid biosynthesis in alfalfa

The potential health benefits of dietary isoflavones have generated considerable interest in engineering the synthesis of these phytoestrogens into plants. Genistein glucoside production (up to 50 nmol g(-1) fresh weight) was engineered in alfalfa (Medicago sativa) leaves by constitutive expression of isoflavone synthase from Medicago truncatula (MtIFS1). Glucosides of biochanin A (4'-O-methylgenistein) and pratensein (3'-hydroxybiochanin A) also accumulated. Although MtIFS1 was highly expressed in all organs examined, genistein accumulation was limited to leaves. MtIFS1-expressing lines accumulated several additional isoflavones, including formononetin and daidzein, in response to UV-B or Phoma medicaginis, whereas the chalcone and flavanone precursors of these compounds accumulated in control lines. Enhanced accumulation of the phytoalexin medicarpin was observed in P. medicaginis-infected leaves of MtIFS1-expressing plants. Microarray profiling indicated that MtIFS1 expression does not significantly alter global gene expression in the leaves. Our results highlight some of the challenges associated with metabolic engineering of plant natural products, including tissue-specific accumulation, potential for further modification by endogenous enzyme activities (hydroxylation, methylation, and glycosylation), and the differential response of engineered plants to environmental factors.     

Genetic and metabolic engineering of isoflavonoid biosynthesis

Isoflavonoids are a diverse group of secondary metabolites derived from the phenylpropanoid pathway. These compounds are distributed predominantly in leguminous plants and play important roles in plant–environment interactions and human health. Consequently, the biosynthetic pathway of isoflavonoid compounds has been widely elucidated in the past decades. Up to now, most of the structural genes and some of the regulatory genes involved in this pathway have been isolated and well characterized. Nowadays, the protective effects of the legume isoflavonoids against hormone dependent cancers, cardiovascular disease, osteoporosis, and menopausal symptoms have generated considerable interest within the genetic and metabolic engineering fields to enhance the dietary intake of these compounds for disease prevention. Subsequently, there are some great progresses in genetic and metabolic engineering to improve their yields in leguminous and non-leguminous plants and/or microorganisms. Because of the field of flavonoid biosynthesis has been reviewed fairly extensively in the past, this review concentrates on the more recent development in the isoflavonoid branch of phenylpropanoid pathway, including gene isolation and characterization. In addition, we describe the state-of-the-art research with respect to genetic and metabolic engineering of isoflavonoid biosynthesis.     

Structure and function of enzymes in heme biosynthesis

Tetrapyrroles like hemes, chlorophylls, and cobalamin are complex macrocycles which play essential roles in almost all living organisms. Heme serves as prosthetic group of many proteins involved in fundamental biological processes like respiration, photosynthesis, and the metabolism and transport of oxygen. Further, enzymes such as catalases, peroxidases, or cytochromes P450 rely on heme as essential cofactors. Heme is synthesized in most organisms via a highly conserved biosynthetic route. In humans, defects in heme biosynthesis lead to severe metabolic disorders called porphyrias. The elucidation of the 3D structures for all heme biosynthetic enzymes over the last decade provided new insights into their function and elucidated the structural basis of many known diseases. In terms of structure and function several rather unique proteins were revealed such as the V‐shaped glutamyl‐tRNA reductase, the dipyrromethane cofactor containing porphobilinogen deaminase, or the “Radical SAM enzyme” coproporphyrinogen III dehydrogenase. This review summarizes the current understanding of the structure–function relationship for all heme biosynthetic enzymes and their potential interactions in the cell.     

Structure,function, and biosynthesis of nickel‐dependent enzymes

Nickel enzymes, present in archaea, bacteria, plants, and primitive eukaryotes are divided into redox and nonredox enzymes and play key functions in diverse metabolic processes, such as energy metabolism and virulence. They catalyze various reactions by using active sites of diverse complexities, such as mononuclear nickel in Ni‐superoxide dismutase, glyoxylase I and acireductone dioxygenase, dinuclear nickel in urease, heteronuclear metalloclusters in [NiFe]‐carbon monoxide dehydrogenase, acetyl‐CoA decarbonylase/synthase and [NiFe]‐hydrogenase, and even more complex cofactors in methyl‐CoM reductase and lactate racemase. The presence of metalloenzymes in a cell necessitates a tight regulation of metal homeostasis, in order to maintain the appropriate intracellular concentration of nickel while avoiding its toxicity. As well, the biosynthesis and insertion of nickel active sites often require specific and elaborated maturation pathways, allowing the correct metal to be delivered and incorporated into the target enzyme. In this review, the phylogenetic distribution of nickel enzymes will be briefly described. Their tridimensional structures as well as the complexity of their active sites will be discussed. In view of the latest findings on these enzymes, a special focus will be put on the biosynthesis of their active sites and nickel activation of apo‐enzymes.     

Structure and function of enzymes involved in the biosynthesis of phenylpropanoids

As a major component of plant specialized metabolism, phenylpropanoid biosynthetic pathways provide anthocyanins for pigmentation, flavonoids such as flavones for protection against UV photodamage, various flavonoid and isoflavonoid inducers of Rhizobium nodulation genes, polymeric lignin for structural support and assorted antimicrobial phytoalexins. As constituents of plant-rich diets and an assortment of herbal medicinal agents, the phenylpropanoids exhibit measurable cancer chemopreventive, antimitotic, estrogenic, antimalarial, antioxidant and antiasthmatic activities. The health benefits of consuming red wine, which contains significant amounts of 3,4',5-trihydroxystilbene (resveratrol) and other phenylpropanoids, highlight the increasing awareness in the medical community and the public at large as to the potential dietary importance of these plant derived compounds. As recently as a decade ago, little was known about the three-dimensional structure of the enzymes involved in these highly branched biosynthetic pathways. Ten years ago, we initiated X-ray crystallographic analyses of key enzymes of this pathway, complemented by biochemical and enzyme engineering studies. We first investigated chalcone synthase (CHS), the entry point of the flavonoid pathway, and its close relative stilbene synthase (STS). Work soon followed on the O-methyl transferases (OMTs) involved in modifications of chalcone, isoflavonoids and metabolic precursors of lignin. More recently, our groups and others have extended the range of phenylpropanoid pathway structural investigations to include the upstream enzymes responsible for the initial recruitment of phenylalanine and tyrosine, as well as a number of reductases, acyltransferases and ancillary tailoring enzymes of phenylpropanoid-derived metabolites. These structure-function studies collectively provide a comprehensive view of an important aspect of phenylpropanoid metabolism. More specifically, these atomic resolution insights into the architecture and mechanistic underpinnings of phenylpropanoid metabolizing enzymes contribute to our understanding of the emergence and on-going evolution of specialized phenylpropanoid products, and underscore the molecular basis of metabolic biodiversity at the chemical level. Finally, the detailed knowledge of the structure, function and evolution of these enzymes of specialized metabolism provide a set of experimental templates for the enzyme and metabolic engineering of production platforms for diverse novel compounds with desirable dietary and medicinal properties.     

Structure,biosynthesis, and function of salivary mucins

The glandular secretions of the oral cavity lining the underlying buccal mucosa are highly specialized fluids which provide lubrication, prevent mechanical damage, protect efficiently against viral and bacterial infections, and promote the clearance of external pollutants. This mucus blanket contains large glycoproteins termed mucins which contribute greatly to the viscoelastic nature of saliva and affect its complex physiological activity. The protein core of mucins consists of repetitive sequences, rich inO-glycosylated serine and threonine, and containing many helix-breaking proline residues. These features account for the extended, somewhat rigid structure of the molecule, a high hydrodynamic volume, its high buoyant density, and high viscosity. The oligosaccharide moiety of salivary mucins accounts for up to 85% of their weight. The oligosaccharide side chains exhibit an astonishing structural diversity. The isolation, composition, structure, molecular characteristics, and functional relevance of salivary mucins and their constituents is discussed in relation to recent advancements in biochemistry and molecular biology.Abbreviations: Abbreviations follow the Rules of Carbohydrate Nomenclature of the American Chemical Society, Carbohydrate Division. All sugars are of thed configuration, exceptl-fucose     

天然生育酚的结构、生物合成和功能

生育酚是一类重要的脂溶性抗氧化荆,天然生育酚有α-、β-、γ-、δ—4种异构体,主要是以尿黑酸为前体在高等植物的叶绿体内膜上合成的。生育酚的主要功能是抗氧化作用,被广泛地应用于制药和食品工业中,开发和提取天然生育酚已引起关注与重视。     

Cytochrome f: Structure,function and biosynthesis

Cytochrome f is an intrinsic membrane component of the cytochrome bf complex, transferring electrons from the Rieske FeS protein to plastocyanin in the thylakoid lumen. The protein is held in the thylakoid membrane by a single transmembrane span located near its C-terminus with a globular hydrophilic domain extending into the lumen. The globular domain of the turnip protein has recently been crystallised, offering the prospect of a detailed three-dimensional structure. Reaction with plastocyanin involves localised positive charges on cytochrome f interacting with the acidic patch on plastocyanin and electron transfer via the surface-exposed tyrosine residue (Tyr83) of plastocyanin. Apocytochrome f is encoded in the chloroplast genome and is synthesised with an N-terminal presequence which targets the protein to the thylakoid membrane. The synthesis of cytochrome f is coordinated with the synthesis of the other subunits of the cytochrome bf complex.     

Structure,function, and biosynthesis of thiazole/oxazole-modified microcins

A great variety of prokaryotic ribosomally synthesized and posttranslationally modified peptides (RiPPs) were predicted and identified experimentally owing to recent advances in large-scale genome sequencing and data analysis. Thiazole/oxazole-modified microcins (TOMMs) are a group of RiPPs that have characteristic thiazole and oxazole heterocycles derived from cysteine and serine residues. The review summarizes the available data on the classification, structure, and biosynthesis of TOMMs and discusses their biological activity and potential use in medicine.     

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.     

细胞色素P450酶的结构、功能与应用研究进展

细胞色素P450 (cytochrome P450,CYP)酶是广泛存在于微生物、动植物及人体中与膜结合的血红蛋白类酶,具有氧化、环氧化、羟化、去甲基化等多种生物催化活性。CYP酶在药物、类固醇、脂溶性维生素和许多其他类型化学物质的代谢中具有重要作用,其在异源物质的解毒、药物相互作用和内分泌功能等领域的研究是热点问题。本综述对CYP的结构、功能、临床应用与开发前景进行了概述,并对其最新的研究现状和发展前景进行探讨。     

Crystal structure of chalcone synthase,a key enzyme for isoflavonoid biosynthesis in soybean

Isoflavonoid is one of the groups of flavonoids that play pivotal roles in the survival of land plants. Chalcone synthase (CHS), the first enzyme of the isoflavonoid biosynthetic pathway, catalyzes the formation of a common isoflavonoid precursor. We have previously reported that an isozyme of soybean CHS (termed GmCHS1) is a key component of the isoflavonoid metabolon, a protein complex to enhance efficiency of isoflavonoid production. Here, we determined the crystal structure of GmCHS1 as a first step of understanding the metabolon structure, as well as to better understand the catalytic mechanism of GmCHS1.     

Structure, biosynthesis and possible function of tunichromes and related compounds

Several species of ascidians (phylum Chordata, subphylum Urochordata) contain a group of oligopeptides called "tunichromes" in their blood cells. These peptides have been implicated in (a) metal chelation and accumulation/sequestration of vanadium or iron; (b) crosslinking of structural fibers in tunic formation, (c) wound healing and (d) defense reactions. However, their biosynthesis, metabolism, and biological function remain largely un-elucidated due to their extreme instability and high reactivity. Tunichromes and related compounds uniquely possess dehydrodopamine moieties, all originating from post-translational modification of peptidyl tyrosine. It is conceivable that the presence of such novel post-translationally modified groups provide attributes that are crucial for their biological roles. Therefore, we examined the chemistry and reactivity of tunichromes in light of the available knowledge of the biochemistry of simple monomeric dehydro-N-acyldopamine units. Based on the reactivity of such simple compounds, the potential biological activities of tunichromes are predicted. Their possible biosynthetic route from peptidyl tyrosine is critically evaluated to provide a better basis for unraveling their biological functions. Prevalence of dehydro-N-acyldopamine units in different tunichromes, some marine antibiotic compounds, insect cuticular sclerotizing precursors and some bioadhesive marine proteins may aid in the de novo design of unique biomaterials with potential antibiotic/adhesive properties.     

Gellan gum biosynthesis in Sphingomonas paucimobilis ATCC 31461: genes,enzymes and exopolysaccharide production engineering

The commercial gelling agent, gellan, is an extracellular polysaccharide (EPS) produced by Sphingomonas paucimobilis ATCC 31461. In recent years, significant progress in understanding the relationship between gellan structure and properties and elucidation of the biosynthesis and engineering of this recent product of biotechnology has been made. This review focuses on recent advances in this field. Emphasis is given to identification and characterization of genes and enzymes involved, or predicted to be involved, in the gellan biosynthetic pathway, at the level of synthesis of sugar-activated precursors, of the repeat unit assembly and of gellan polymerization and export. Identification of several genes, biochemical characterization of the encoded enzymes and elucidation of crucial steps of the gellan pathway indicate that possibilities now exist for exerting control over gellan production at any of the three levels of its biosynthesis. However, a better knowledge of the poorly understood steps and of the bottlenecks and regulation of the pathway, the characterization of the composition, structure and functional properties of gellan-like polymers produced either by the industrial strain under different culture conditions or by mutants are still required for eventual success of the metabolic engineering of gellan production. Journal of Industrial Microbiology & Biotechnology (2002) 29, 170–176 doi:10.1038/sj.jim.7000266 Received 11 February 2002/ Accepted in revised form 09 April 2002     

Structure, biosynthesis, and function of asparagine-linked glycans on plant glycoproteins

In plants, glycoproteins with asparagine-linked glycans (oligosaccharides) are found in vacuoles, in the extracellular space or matrix, and associated with the endo-membrane system (endoplasmic reticulum, Golgi apparatus, plasma membrane, tonoplast). These glycans are of the high-mannose type, with a structure identical to that found in other organisms (mammals, yeast), or of the complex type with a β1–2 linked xylosyl residue not found in mammalian complex glycans. Asparagine-linked glycans play multiple roles by modifying the physicochemical properties of the polypeptides to which they are attached.     

Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering

Details of the recently elucidated biosynthetic pathways of caffeine and related purine alkaloids are reviewed. The main caffeine biosynthetic pathway is a sequence consisting of xanthosine-->7-methylxanthosine-->7-methylxanthine-->theobromine-->caffeine. Genes encoding N-methyltransferases involved in three of these four reactions have been isolated and the molecular structure of N-methyltransferases investigated. Pathways for the catabolism of caffeine have also been studied, although there are currently no reports of enzymatic and genetic studies having been successfully carried out. Metabolism of purine alkaloids in species including Camellia, Coffea, Theobroma and Ilex plants is summarised, and evidence for the involvement of caffeine in chemical defense and allelopathy is discussed. Finally, information is presented on metabolic engineering that has produced coffee seedlings with reduced caffeine content, and transgenic caffeine-producing tobacco plants with enhanced disease resistance.