Cloning, nucleotide sequence, and heterologous expression of the biosynthetic gene cluster for R1128, a non-steroidal estrogen receptor antagonist. Insights into an unusual priming mechanism

R1128 substances are anthraquinone natural products that were previously reported as non-steroidal estrogen receptor antagonists with in vitro and in vivo potency approaching that of tamoxifen. From a biosynthetic viewpoint, these polyketides possess structurally interesting features such as an unusual primer unit that are absent in the well studied anthracyclic and tetracyclic natural products. The entire R1128 gene cluster was cloned and expressed in Streptomyces lividans, a genetically well developed heterologous host. In addition to R1128C, a novel optically active natural product, designated HU235, was isolated. Nucleotide sequence analysis of the biosynthetic gene cluster revealed genes encoding two ketosynthases, a chain length factor, an acyl transferase, three acetyl-CoA carboxylase subunits, two cyclases, two oxygenases, an amidase, and remarkably, two acyl carrier proteins. Feeding studies indicate that the unusual 4-methylvaleryl side chain of R1128C is derived from valine. Together with the absence of a dedicated ketoreductase, dehydratase, or enoylreductase within the R1128 gene cluster, this suggests a functional link between fatty acid biosynthesis and R1128 biosynthesis in the engineered host. Specifically, we propose that the R1128 synthase recruits four subunits from the endogenous fatty acid synthase during the biosynthesis of this family of pharmacologically significant natural products.     

Structure and function of the RedJ protein, a thioesterase from the prodiginine biosynthetic pathway in Streptomyces coelicolor

Prodiginines are a class of red-pigmented natural products with immunosuppressant, anticancer, and antimalarial activities. Recent studies on prodiginine biosynthesis in Streptomyces coelicolor have elucidated the function of many enzymes within the pathway. However, the function of RedJ, which was predicted to be an editing thioesterase based on sequence similarity, is unknown. We report here the genetic, biochemical, and structural characterization of the redJ gene product. Deletion of redJ in S. coelicolor leads to a 75% decrease in prodiginine production, demonstrating its importance for prodiginine biosynthesis. RedJ exhibits thioesterase activity with selectivity for substrates having long acyl chains and lacking a β-carboxyl substituent. The thioesterase has 1000-fold greater catalytic efficiency with substrates linked to an acyl carrier protein (ACP) than with the corresponding CoA thioester substrates. Also, RedJ strongly discriminates against the streptomycete ACP of fatty acid biosynthesis in preference to RedQ, an ACP of the prodiginine pathway. The 2.12 Å resolution crystal structure of RedJ provides insights into the molecular basis for the observed substrate selectivity. A hydrophobic pocket in the active site chamber is positioned to bind long acyl chains, as suggested by a long-chain ligand from the crystallization solution bound in this pocket. The accessibility of the active site is controlled by the position of a highly flexible entrance flap. These data combined with previous studies of prodiginine biosynthesis in S. coelicolor support a novel role for RedJ in facilitating transfer of a dodecanoyl chain from one acyl carrier protein to another en route to the key biosynthetic intermediate 2-undecylpyrrole.     

Enzymatic assembly of epothilones: the EpoC subunit and reconstitution of the EpoA-ACP/B/C polyketide and nonribosomal peptide interfaces

The biosynthesis of epothilones, a family of hybrid polyketide (PK)/nonribosomal peptide (NRP) antitumor agents, provides an ideal system to study a hybrid PK/NRP natural product with significant biomedical value. Here the third enzyme involved in epothilone production, the five domain 195 kDa polyketide synthase (PKS) EpoC protein, has been expressed and purified from Escherichia coli. EpoC was combined with the first two enzymes of the epothilone biosynthesis pathway, the acyl carrier protein (ACP) domain of EpoA and EpoB, to reconstitute the early steps in epothilone biosynthesis. The acyltransferase (AT) domain of EpoC transfers the methylmalonyl moiety from methylmalonyl-CoA to the holo HS-acyl carrier protein (ACP) in an autoacylation reaction. The ketosynthase (KS) domain of EpoC decarboxylates the methylmalonyl-S-EpoC acyl enzyme to generate the carbon nucleophile that reacts with methylthiazolylcarboxyl-S-EpoB. The resulting condensation product can be reduced in the presence of NADPH by the ketoreductase (KR) domain of EpoC and then dehydrated by the dehydratase (DH) domain to produce the methylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediate that serves as the acyl donor for subsequent elongation of the epothilone chain. The acetyl-CoA donor can be replaced with propionyl-CoA, isobutyryl-CoA, and benzoyl-CoA and the acyl chains accepted by both EpoB and EpoC subunits to produce ethyl-, isopropyl-, and phenylthiazolylmethylacrylyl-S-EpoC acyl enzyme intermediates, suggesting that future combinatorial biosynthetic variations in epothilone assembly may be feasible. These results demonstrate in vitro reconstitution of both the PKS/NRPS interface (EpoA-ACP/B) and the NRPS/PKS interface (EpoB/C) in the assembly line for this antitumor natural product.     

Carrier protein structure and recognition in polyketide and nonribosomal peptide biosynthesis

Carrier proteins, 80-100 residues in length, serve as information-rich platforms to present growing acyl and peptidyl chains as covalently tethered phosphopantetheinyl-thioester intermediates during the biosynthesis of fatty acid, polyketide, and nonribosomal natural products. Carrier proteins are recognized both in cis and in trans by partner catalytic domains that effect chain-elongating condensations, redox adjustments, other tailoring steps, and finally kinetically controlled disconnection and release of the mature natural product. Dissection of regions of carrier proteins that are specifically recognized by upstream and downstream catalytic partner proteins is deciphering the logic for multiprotein assembly line construction of these large classes of natural products.     

Biosynthesis of triphosphoribosyl-dephospho-coenzyme A, the precursor of the prosthetic group of malonate decarboxylase

Malonate decarboxylase from Klebsiella pneumoniae consists of four subunits MdcA, D, E, and C and catalyzes the cleavage of malonate to acetate and CO(2). The smallest subunit MdcC is an acyl carrier protein to which acetyl and malonyl thioester residues are bound via a 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group and turn over during the catalytic mechanism. We report here on the biosynthesis of holo acyl carrier protein from the unmodified apoprotein. The prosthetic group biosynthesis starts with the MdcB-catalyzed condensation of dephospho-CoA with ATP to 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA. In this reaction, a new alpha (1' ' --> 2') glycosidic bond between the two ribosyl moieties is formed, and thereby, the adenine moiety of ATP is displaced. MdcB therefore is an ATP:dephospho-CoA 5'-triphosphoribosyl transferase. The second protein involved in holo ACP synthesis is MdcG. This enzyme forms a strong complex with the 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA prosthetic group precursor. This complex, called MdcG(i), is readily separated from free MdcG by native polyacrylamide gel electrophoresis. Upon incubation of MdcG(i) with apo acyl carrier protein, holo acyl carrier protein is synthesized by forming the phosphodiester bond between the 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group and serine 25 of the protein. MdcG corresponds to a 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA:apo ACP 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA transferase. In absence of the prosthetic group precursor, MdcG catalyzes at a low rate the adenylylation of apo acyl carrier protein using ATP as substrate. The adenylyl ACP thus formed is an unphysiological side product and is not involved in the biosynthesis of holo ACP. The 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA precursor of the prosthetic group has been purified and its identity confirmed by mass spectrometry and enzymatic analysis.     

Building-block selectivity of polyketide synthases

For the past decade, polyketide synthases have presented an exciting paradigm for the controlled manipulation of complex natural product structure. These multifunctional enzymes catalyze the biosynthesis of polyketide natural products by stepwise condensation and modification of metabolically derived building blocks. In particular, regioselective modification of polyketide structure is possible by alterations in either intracellular acyl-CoA pools or, more commonly, by manipulation of acyl transferases that act as the primary gatekeepers for building blocks.     

Structures and Properties of Gellan Polymers Produced by Sphingomonas paucimobilis ATCC 31461 from Lactose Compared with Those Produced from Glucose and from Cheese Whey

The dairy industry produces large quantities of whey as a by-product of cheese production and is increasingly looking for new ways to utilize this waste product. Gellan gum is reliably produced by Sphingomonas paucimobilis in growth media containing lactose, a significant component of cheese whey, as a carbon source. We studied and compared polysaccharide biosynthesis by S. paucimobilis ATCC 31461 in media containing glucose, lactose (5 to 30 g/liter), and sweet cheese whey. We found that altering the growth medium can markedly affect the polysaccharide yield, acyl substitution level, polymer rheological properties, and susceptibility to degradation. Depression of gellan production from lactose compared with gellan production from glucose (approximately 30%) did not appear to occur at the level of synthesis of sugar nucleotides, which are the donors of monomers used for biosynthesis of the repetitive tetrasaccharide unit of gellan. The lactose-derived biopolymer had the highest total acyl content; the glucose- and whey-derived gellans had similar total acyl contents but differed markedly in their acetate and glycerate levels. Rheological studies revealed how the functionality of a gellan polysaccharide is affected by changes in the acyl substitution.     

Current status, strategies, and potential for the metabolic engineering of heterologous polyketides in Escherichia coli

Heterologous natural product biosynthesis has emerged as a strategy to produce medicinal compounds that pose challenges to conventional production routes. Polyketide compounds, an important class of natural products with wide-ranging therapeutic value, have been heterologously produced through Escherichia coli, presenting new opportunities to realize the medicinal potential of polyketide natural products. However, current production levels are often suboptimal when compared to native strain producers or heterologous theoretical yields. This problem provides an excellent opportunity to apply and further develop current metabolic engineering tools.     

色氨酸, 天然产物生物合成中的重要结构单元

氨基酸作为生物体内组成生命物质的小分子化合物, 在天然产物生物合成中扮演了非常重要的作用。色氨酸含有一个独特的吲哚环, 相对复杂的吲哚环平面结构使得色氨酸相比其他氨基酸具有更多的修饰空间。在微生物天然产物生物合成研究中, 色氨酸及其衍生物经常作为组成模块参与到天然产物的生物合成中, 本文概述了色氨酸几种不同的生物修饰方式, 包括烷基化修饰、卤化修饰、羟基化修饰、以及吲哚环的开环重排反应等。分析并总结色氨酸在天然产物生物合成中的作用可以增加我们对天然产物结构多样性的认识和推动天然产物生物合成机制的研究。     

The significance of light activation of enzymes during the induction phase of photosynthesis in isolated chloroplasts

The key reaction of flavonoid biosynthesis, the condensation of the acyl residues from one molecule of 4-coumaroyl-CoA and three molecules of malonyl-CoA, has previously been assumed to be catalyzed by a “flavanone synthase.” Results are presented here which indicate that not the flavanone but the isomeric chalcone is the immediate product of the synthase reaction. The new term “chalcone synthase” is therefore suggested for the enzyme.     

Bio-production of Baccatin III,an Important Precursor of Paclitaxel by a Cost-Effective Approach

Natural production of anti-cancer drug taxol from Taxus has proved to be environmentally unsustainable and economically unfeasible. Currently, bioengineering the biosynthetic pathway of taxol is an attractive alternative production approach. 10-deacetylbaccatin III-10-O-acetyl transferase (DBAT) was previously characterized as an acyltransferase, using 10-deacetylbaccatin III (10-DAB) and acetyl CoA as natural substrates, to form baccatin III in the taxol biosynthesis. Here, we report that other than the natural acetyl CoA (Ac-CoA) substrate, DBAT can also utilize vinyl acetate (VA), which is commercially available at very low cost, acylate quickly and irreversibly, as acetyl donor in the acyl transfer reaction to produce baccatin III. Furthermore, mutants were prepared via a semi-rational design in this work. A double mutant, I43S/D390R was constructed to combine the positive effects of the different single mutations on catalytic activity, and its catalytic efficiency towards 10-DAB and VA was successfully improved by 3.30-fold, compared to that of wild-type DBAT, while 2.99-fold higher than the catalytic efficiency of WT DBAT towards 10-DAB and Ac-CoA. These findings can provide a promising economically and environmentally friendly method for exploring novel acyl donors to engineer natural product pathways.     

Unlocking the potential of natural products in drug discovery

The natural product specialized metabolites produced by microbes and plants are the backbone of our current drugs. Despite their historical importance, few pharmaceutical companies currently emphasize their exploitation in new drug discovery and instead favour synthetic compounds as more tractable alternatives. Ironically, we are in a Golden Age of understanding of natural product biosynthesis, biochemistry and engineering. These advances have the potential to usher in a new era of natural product exploration and development taking full advantage of the unique and favourable properties of natural products compounds in drug discovery.     

Dimeric structure of the six-domain VibF subunit of vibriobactin synthetase: mutant domain activity regain and ultracentrifugation studies

Nonribosomal peptide synthetases (NRPS), fatty acid synthases (FAS), and polyketide sythases (PKS) are multimodular enzymatic assembly lines utilized in natural product biosynthesis. Previous data on FAS and PKS subunits have indicated that they are homodimers and that some of their catalytic functions can work in trans. When NRPS assembly lines have been probed for comparable formation of stable oligomers, no evidence had been forthcoming that species other than monomer forms were active. In this work we focus on the six-domain (Cy1-Cy2-A-C1-PCP-C2) enzyme VibF from the vibriobactin synthetase assembly line, which contains three other proteins, VibB, VibE, and VibH, that--when purified and mixed with VibF and the substrates ATP, threonine, 2,3-dihydroxybenzoate (DHB), and norspermidine--produce the iron chelator vibriobactin. Using a deletion of the Cy1 domain and separate inactivating mutations in the Cy2, A, PCP, and C2 domains of VibF, we report regain of catalytic activity upon mutant protein mixing that argues for heterodimer formation, stable for hundreds to thousands of catalytic cycles, with acyl chain processing and transfer around blocked domains. Ultracentrifugation data likewise confirm a dimeric structure for VibF and establish that domains within NRPS dimeric modules can act on acyl chains in trans. The results described here are the first indication for an NRPS subunit that homodimerization can occur and that there is a continuum of functional oligomerization states between monomers and dimers in nonribosomal peptide synthetases.     

植物丝氨酸羧肽酶样酰基转移酶的结构、功能和应用

植物丝氨酸羧肽酶样酰基转移酶与丝氨酸羧肽酶相比具有相似的结构特点和很高的同源性,能够催化酰基葡萄糖酯的酰基转移反应,参与植物次生代谢产物的酰基化修饰,丰富天然产物结构多样性,改善化合物水溶性、稳定性等理化性质.本文重点介绍植物来源丝氨酸羧肽酶样酰基转移酶家族的结构特点、催化机制、功能鉴定及其生物催化应用等方面的研究进展...     

1,4-Naphthoquinone,an intermediate in juglone (5-hydroxy-1,4-naphthoquinone) biosynthesis

Evidence is presented which shows that 1,4-naphthoquinone, a new natural product, is involved in the biosynthesis ofjuglone in Juglans regia.     

Chemical mechanism and substrate specificity of RhlI, an acylhomoserine lactone synthase from Pseudomonas aeruginosa

The enzyme RhlI catalyzes the formation of N-butyrylhomoserine lactone from S-adenosylmethionine and N-butyrylacyl carrier protein. N-Butyrylhomoserine lactone serves as a quorum-sensing signal molecule in Pseudomonas aeruginosa, and is implicated in the regulation of many processes involved in bacterial virulence and infectivity. The P. aeruginosa genome contains three genes encoding acyl carrier proteins. We have cloned all three genes, expressed the acyl carrier proteins, and characterized each as a substrate for RhlI. A continuous, spectrophotometric assay was developed to facilitate kinetic and mechanistic studies of RhlI. Acp1, which has not been characterized previously, was a good substrate for RhlI, with a K(m) of 7 microM; the reaction proceeded with a k(cat) value of 0.35 s(-1). AcpP, which supports fatty acid biosynthesis, was also a good substrate in the RhlI reaction, where k(cat) was 0.46 s(-1), and the K(m) for AcpP was 6 microM. The third acyl carrier protein, Acp3, was a poor substrate for RhlI, with a K(m) of 280 microM; k(cat) was 0.03 s(-1). Taken together with microarray data from the literature which show that expression of the gene encoding Acp1 is under the control of the quorum-sensing system, our data suggest that Acp1 is likely to be the substrate for RhlI in vivo. Isotope labeling studies were conducted to investigate the chemical mechanism of the RhlI-catalyzed lactonization reaction. Solvent deuterons were not incorporated into product, which implicates a direct attack mechanism in which the carboxylate oxygen of the presumptive N-butyryl-SAM intermediate attacks the methylene carbon adjacent to the sulfonium ion. Alternative mechanisms, in which N-butyrylvinylglycine is formed via elimination of methylthioadenosine, were ruled out on the basis of the observation that RhlI failed to convert authentic N-butyrylvinylglycine to N-butyryl-L-homoserine lactone.     

草菇聚酮合酶编码基因vv-alb的克隆及其表达的初步分析

聚酮化合物（polyketides）是一类庞大的次级代谢家族,聚酮合酶（polyketide synthase,PKS）是介导聚酮化合物生物合成的关键酶。通过巢氏简并PCR与染色体步行的方法,获得了草菇中的编码PKS的基因vv-alb的全长序列,并通过荧光实时定量RT-PCR方法对vv-alb基因在草菇不同生长阶段与不同部位的表达情况进行了初步分析,为进一步研究PKS在草菇和其他食用真菌生物代谢过程中的作用奠定了一定的基础。     

Structural and biochemical characterization of the salicylyl-acyltranferase SsfX3 from a tetracycline biosynthetic pathway

SsfX3 is a GDSL family acyltransferase that transfers salicylate to the C-4 hydroxyl of a tetracycline intermediate in the penultimate step during biosynthesis of the anticancer natural product SF2575. The C-4 salicylate takes the place of the more common C-4 dimethylamine functionality, making SsfX3 the first acyltransferase identified to act on a tetracycline substrate. The crystal structure of SsfX3 was determined at 2.5 Å, revealing two distinct domains as follows: an N-terminal β-sandwich domain that resembles a carbohydrate-binding module, and a C-terminal catalytic domain that contains the atypical α/β-hydrolase fold found in the GDSL hydrolase family of enzymes. The active site lies at one end of a large open binding pocket, which is spatially defined by structural elements from both the N- and C-terminal domains. Mutational analysis in the putative substrate binding pocket identified residues from both domains that are important for binding the acyl donor and acceptor. Furthermore, removal of the N-terminal carbohydrate-binding module-like domain rendered the stand-alone α/β-hydrolase domain inactive. The additional noncatalytic module is therefore proposed to be required to define the binding pocket and provide sufficient interactions with the spatially extended tetracyclic substrate. SsfX3 was also demonstrated to accept a variety of non-native acyl groups. This relaxed substrate specificity toward the acyl donor allowed the chemoenzymatic biosynthesis of C-4-modified analogs of the immediate precursor to the bioactive SF2575; these were used to assay the structure activity relationships at the C-4 position.     

Structural insights into biosynthesis of resorcinolic lipids by a type III polyketide synthase in Neurospora crassa

Microbial type III polyketide synthases (PKSs) have revealed remarkable mechanistic as well as functional versatility. Recently, a type III PKS homolog from Azotobacter has been implicated in the biosynthesis of resorcinolic lipids, thus adding a new functional significance to this class of proteins. Here, we report the structural and mutational investigations of a novel type III PKS protein from Neurospora crassa involved in the biosynthesis of resorcinolic metabolites by utilizing long chain fatty acyl-CoAs. The structure revealed a long hydrophobic tunnel responsible for its fatty acyl chain length specificity resembling that of PKS18, a mycobacterial type III PKS. Structure-based mutational studies to block the tunnel not only altered the fatty acyl chain specificity but also resulted in change of cyclization pattern affecting the product profile. This first structural characterization of a resorcinolic lipid synthase provides insights into the coordinated functioning of cyclization and a substrate-binding pocket, which shows mechanistic intricacy underlying type III PKS catalysis.     

The enzymology of combinatorial biosynthesis

Combinatorial biosynthesis involves the genetic manipulation of natural product biosynthetic enzymes to produce potential new drug candidates that would otherwise be difficult to obtain. In either a theoretical or practical sense, the number of combinations possible from different types of natural product pathways ranges widely. Enzymes that have been the most amenable to this technology synthesize the polyketides, nonribosomal peptides, and hybrids of the two. The number of polyketide or peptide natural products theoretically possible is huge, but considerable work remains before these large numbers can be realized. Nevertheless, many analogs have been created by this technology, providing useful structure-activity relationship data and leading to a few compounds that may reach the clinic in the next few years. In this review the focus is on recent advances in our understanding of how different enzymes for natural product biosynthesis can be used successfully in this technology.