Catalytic relationships between type I and type II iterative polyketide synthases: The Aspergillus parasiticus norsolorinic acid synthase

Norsolorinic acid synthase (NSAS) is a type I iterative polyketide synthase that occurs in the filamentous fungus Aspergillus parasiticus. PCR was used to clone fragments of NSAS corresponding to the acyl carrier protein (ACP), acyl transferase (AT) and beta-ketoacyl-ACP synthase (KS) catalytic domains. Expression of these gene fragments in Escherichia coli led to the production of soluble ACP and AT proteins. Coexpression of ACP with E. coli holo-ACP synthase (ACPS) let to production of NSAS holo-ACP, which could also be formed in vitro by using Streptomyces coelicolor ACPS. Analysis by mass spectrometry showed that, as with other type I carrier proteins, self-malonylation is not observed in the presence of malonyl CoA alone. However, the NSAS holo-ACP serves as substrate for S. coelicolor MCAT, S. coelicolor actinorhodin holo-ACP and NSAS AT domain-catalysed malonate transfer from malonyl CoA. The AT domain could transfer malonate from malonyl CoA to NSAS holo-ACP, but not hexanoate or acetate from either the cognate CoA or FAS ACP species to NSAS holo-ACP. The NSAS holo-ACP was also active in actinorhodin minimal PKS assays, but only in the presence of exogenous malonyl transferases.     

Demonstration of starter unit interprotein transfer from a Fatty Acid synthase to a multidomain, nonreducing polyketide synthase

The pathway for substrate transacylation between a fungal type I fatty acid synthase (FAS) and a nonreducing polyketide synthase (NR-PKS) was determined by in vitro reconstitution of dissected domains. System kinetics were influenced by domain dissections, and the FAS phosphopantetheinyl transferase (PPT) monodomain exhibited coenzyme A selectivity for the post-translational activation of the FAS acyl carrier protein (ACP).     

In Vitro Reconstitution of a PKS Pathway for the Biosynthesis of Galbonolides in Streptomyces sp. LZ35

The galbonolides are 14‐membered macrolide antibiotics with a macrocyclic backbone similar to that of erythromycins. Galbonolides exhibit broad‐spectrum antifungal activities. Retro‐biosynthetic analysis suggests that the backbone of galbonolides is assembled by a type I modular polyketide synthase (PKS). Unexpectedly, the galbonolide biosynthetic gene cluster, gbn, in Streptomyces sp. LZ35 encodes a hybrid fatty acid synthase (FAS)‐PKS pathway. In vitro reconstitution revealed the functions of GbnA (an AT‐ACP didomain protein), GbnC (a FabH‐like enzyme), and GbnB (a novel multidomain PKS module without AT and ACP domains) responsible for assembling the backbone of galbonolides, respectively. To our knowledge, this study is the first biochemical characterization of a hybrid FAS‐PKS pathway for the biosynthesis of 14‐membered macrolides. The identification of this pathway provides insights into the evolution of PKSs and could facilitate the design of modular pools for synthetic biology.     

A polylinker approach to reductive loop swaps in modular polyketide synthases

Multiple versions of the DEBS 1-TE gene, which encodes a truncated bimodular polyketide synthase (PKS) derived from the erythromycin-producing PKS, were created by replacing the DNA encoding the ketoreductase (KR) domain in the second extension module by either of two synthetic oligonucleotide linkers. This made available a total of nine unique restriction sites for engineering. The DNA for donor "reductive loops," which are sets of contiguous domains comprising either KR or KR and dehydratase (DH), or KR, DH and enoylreductase (ER) domains, was cloned from selected modules of five natural PKS multienzymes and spliced into module 2 of DEBS 1-TE using alternative polylinker sites. The resulting hybrid PKSs were tested for triketide production in vivo. Most of the hybrid multienzymes were active, vindicating the treatment of the reductive loop as a single structural unit, but yields were dependent on the restriction sites used. Further, different donor reductive loops worked optimally with different splice sites. For those reductive loops comprising DH, ER and KR domains, premature TE-catalysed release of partially reduced intermediates was sometimes seen, which provided further insight into the overall stereochemistry of reduction in those modules. Analysis of loops containing KR only, which should generate stereocentres at both C-2 and C-3, revealed that the 3-hydroxy configuration (but not the 2-methyl configuration) could be altered by appropriate choice of a donor loop. The successful swapping of reductive loops provides an interesting parallel to a recently suggested pathway for the natural evolution of modular PKSs by recombination.     

In vivo mutational analysis of the mupirocin gene cluster reveals labile points in the biosynthetic pathway: the "leaky hosepipe" mechanism

A common feature of the mupirocin and other gene clusters of the AT-less polyketide synthase (PKS) family of metabolites is the introduction of carbon branches by a gene cassette that contains a beta-hydroxy-beta-methylglutaryl CoA synthase (HMC) homologue and acyl carrier protein (ACP), ketosynthase (KS) and two crotonase superfamily homologues. In vivo studies of Pseudomonas fluorescens strains in which any of these components have been mutated reveal a common phenotype in which the two major isolable metabolites are the truncated hexaketide mupirocin H and the tetraketide mupiric acid. The structure of the latter has been confirmed by stereoselective synthesis. Mupiric acid is also the major metabolite arising from inactivation of the ketoreductase (KR) domain of module 4 of the modular PKS. A number of other mutations in the tailoring region of the mupirocin gene cluster also result in production of both mupirocin H and mupiric acid. To explain this common phenotype we propose a mechanistic rationale in which both mupirocin H and mupiric acid represent the products of selective and spontaneous release from labile points in the pathway that occur at significant levels when mutations block the pathway either close to or distant from the labile points.     

真菌芳香聚酮合酶研究进展

真菌芳香聚酮化合物是由真菌非还原聚酮合酶（NR-PKS）催化合成的一大类具有生物活性的天然产物。真菌芳香聚酮合酶是一个包含酮基合酶（KS）、酰基载体蛋白（ACP）、起始单元：ACP酰基转移酶（SAT）、丙二酰：ACP酰基转移酶（MAT）、产物模板（PT）和硫酯-环化酶（TE-CLC）6个结构域的巨大蛋白,负责催化真菌芳香聚酮的生物合成。在催化循环中如何选择起始单元,如何催化链的起始与延伸,如何进行中间体的折叠与环化,以及如何控制产物的正确解离,是真菌芳香聚酮合酶催化机制中的核心问题。对这些问题的理解为从真菌中筛选天然产物合成途径和利用组合生物合成手段产生新型活性分子奠定了基础。     

An ACP structural switch: conformational differences between the apo and holo forms of the actinorhodin polyketide synthase acyl carrier protein

The actinorhodin (act) synthase acyl carrier protein (ACP) from Streptomyces coelicolor plays a central role in polyketide biosynthesis. Polyketide intermediates are bound to the free sulfhydryl group of a phosphopantetheine arm that is covalently linked to a conserved serine residue in the holo form of the ACP. The solution NMR structures of both the apo and holo forms of the ACP are reported, which represents the first high resolution comparison of these two forms of an ACP. Ensembles of twenty apo and holo structures were calculated and yielded atomic root mean square deviations of well-ordered backbone atoms to the average coordinates of 0.37 and 0.42 A, respectively. Three restraints defining the protein to the phosphopantetheine interface were identified. Comparison of the apo and holo forms revealed previously undetected conformational changes. Helix III moved towards helix II (contraction of the ACP), and Leu43 on helix II subtly switched from being solvent exposed to forming intramolecular interactions with the newly added phosphopantetheine side chain. Tryptophan fluorescence and S. coelicolor fatty acid synthase (FAS) holo-synthase (ACPS) assays indicated that apo-ACP has a twofold higher affinity (K(d) of 1.1 muM) than holo-ACP (K(d) of 2.1 muM) for ACPS. Site-directed mutagenesis of Leu43 and Asp62 revealed that both mutations affect binding, but have differential affects on modification by ACPS. Leu43 mutations in particular strongly modulate binding affinity for ACPS. Comparison of apo- and holo-ACP structures with known models of the Bacillus subtilis FAS ACP-holo-acyl carrier protein synthase (ACPS) complex suggests that conformational modulation of helix II and III between apo- and holo-ACP could play a role in dissociation of the ACP-ACPS complex.     

In Vitro Investigation of Crosstalk between Fatty Acid and Polyketide Synthases in the Andrimid Biosynthetic Assembly Line

Andrimid (Adm) synthase, which belongs to the type II system of enzymes, produces Adm in Pantoea agglomerans. The adm biosynthetic gene cluster lacks canonical acyltransferases (ATs) to load the malonyl group to acyl carrier proteins (ACPs), thus suggesting that a malonyl‐CoA ACP transacylase (MCAT) from the fatty acid synthase (FAS) complex provides the essential AT activity in Adm biosynthesis. Here we report that an MCAT is essential for catalysis of the transacylation of malonate from malonyl‐CoA to AdmA polyketide synthase (PKS) ACP in vitro. Catalytic self‐malonylation of AdmA (PKS ACP) was not observed in reactions without MCAT, although many type II PKS ACPs are capable of catalyzing self‐acylation. This lack of self‐malonylation was explained by amino acid sequence analysis of the AdmA PKS ACP and the type II PKS ACPs. The results show that MCAT from the organism's FAS complex can provide the missing AT activity in trans, thus suggesting a protein–protein interaction between the fatty acid and polyketide synthases in the Adm assembly line.     

Using Modern Tools To Probe the Structure–Function Relationship of Fatty Acid Synthases

Fatty acid biosynthesis is essential to life and represents one of the most conserved pathways in nature, preserving the same handful of chemical reactions across all species. Recent interest in the molecular details of the de novo fatty acid synthase (FAS) has been heightened by demand for renewable fuels and the emergence of multidrug‐resistant bacterial strains. Central to FAS is the acyl carrier protein (ACP), a protein chaperone that shuttles the growing acyl chain between catalytic enzymes within the FAS. Human efforts to alter fatty acid biosynthesis for oil production, chemical feedstock, or antimicrobial purposes has been met with limited success, due in part to a lack of detailed molecular information behind the ACP–partner protein interactions inherent to the pathway. This review will focus on recently developed tools for the modification of ACP and analysis of protein–protein interactions, such as mechanism‐based crosslinking, and the studies exploiting them. Discussion specific to each enzymatic domain will focus first on mechanism and known inhibitors, followed by available structures and known interactions with ACP. Although significant unknowns remain, new understandings of the intricacies of FAS point to future advances in manipulating this complex molecular factory.     

Covalent linkage mediates communication between ACP and TE domains in modular polyketide synthases

Polyketide natural products such as erythromycin A and epothilone are assembled on multienzyme polyketide synthases (PKSs), which consist of modular sets of protein domains. Within these type I systems, the fidelity of biosynthesis depends on the programmed interaction among the multiple domains within each module, centered around the acyl carrier protein (ACP). A detailed understanding of interdomain communication will therefore be vital for attempts to reprogram these pathways by genetic engineering. We report here that the interaction between a representative ACP domain and its downstream thioesterase (TE) is mediated largely by covalent tethering through a short "linker" region, with only a minor energetic contribution from protein-protein molecular recognition. This finding helps explain in part the empirical observation that TE domains can function out of their normal context in engineered assembly lines, and supports the view that overall PKS architecture may dictate at least a subset of interdomain interactions.     

Modeling multi-component protein-DNA complexes: the role of bending and dimerization in the complex of p53 dimers with DNA

We used molecular modeling to study the optimal conformationof the complex between two p53 DNA-binding domain monomers anda 12 base-pair target DNA sequence. The complex was constructedusing experimental data on the monomer binding conformationand a new approach to deform the target DNA sequence. Combinedwith an internal/helicoidal coordinate model of DNA, this approachenables us to bend the target sequence in a controlled way whilerespecting the contacts formed with each p53 monomer. The resultsshow that the dimeric complex favors DNA bending towards themajor groove at the dimer junction by a value close to experimentalfindings. In contrast to inferences from earlier models, thecalculation of key contributions to the free energy of the complexesindicates a determinant role for DNA in the formation of thecomplex with the dimer of the p53 DNA-binding domains.     

The reductase steps of the type II fatty acid synthase as antimicrobial targets

The increasing of multidrug resistance of clinically important pathogens calls for the development of novel antibiotics with unexploited cellular targets. FA biosynthesis in bacteria is catalyzed by a group of highly conserved proteins known as the type II FA synthase (FAS II) system. Bacteria FAS II organization is distinct from its mammalian counterpart; thus the FAS II pathway offers several unique steps for selective inhibition by antibacterial agents. Some known antibiotics that target the FAS II system include triclosan, isoniazid, and thiolactomycin. Recent years have seen remarkable progress in the understanding of the genetics, biochemistry, and regulation of the FAS II system with the availability of the complete geome, sequence for many bacteria. Crystal structures of the FAS II pathway enzymes have been determined for not only the Escherichia coli model system but also other gram-netative and gram-positive pathogens. The protein structures have greatly facilitated structure-based design of novel inhibitors and the improvement of existing antibacterial agents. This review discusses new developments in the discovery of inhibitors that specifically target the two reductase steps of the FAS II system, β-ketoacyl-acyl carrier potein (ACP) reductase and enoyl-ACP reductase.     

Improvement of palm oil through breeding and biotechnology

The oil palm Elaeis guineensis is the highest oil-yielding crop and has the potential to become the major supplier of both edible oil and renewable industrial feedstock. The oil yield from wild groves is presently less than 0.5 t/ha/y. However, through breeding and selection, the oil yield of commercial plantations could reach as much as 8 t/ha/y. New planting materials also have the capability of better oil yields with high iodine value (IV), slow height increment, and larger kernels. The oil also contains considerable amounts of carotenoids (500–700 ppm), vitamin E (600–1000 ppm), and sterols (250–620 ppm). The oil yield of another oil palm species, E. oleifera, is approximately 0.5 t/ha/y with high contents of carotenoids (700–1500 ppm), vitamin E (700–1500 ppm), and sterols (3500–4000 ppm). The above traits could be improved through breeding and biotechnology. Biotechnological efforts at the Palm Oil Institute of Malaysia are directed toward the production of oil with high IV and high monounsaturated fatty acids for edible purposes and industrial uses. Isolation and manipulation of the genes involved in the biosynthesis of fatty acids are the main focus. The aim is to increase the efficiency of conversion of palmitate (C_16:0) to oleate (C_18:1). Levels of palmitate and oleate are controlled by the enzymes acyl-acyl carrier protein (ACP) thioesterase and β-keto acyl ACP synthase II. The chain termination reactions of C_16:0 and C_18:1 are independent, thus paving the way for the possibility of reducing palmitate levels by switching off the palmitoyl ACP thioesterase gene. Paper presented at the 88th AOCS Annual Meeting & Expo. May 11–14, 1997, Seattle, Washington, USA.     

Probing the Molecular Mechanisms in Copper Amine Oxidases by Generating Heterodimers

For some homodimeric copper amine oxidases (CuAO), there is suggestive evidence of differential activity at the two active sites implying potential cooperativity between the two monomers. To examine this phenomenon for the Arthrobacter globiformis CuAO (AGAO), we purified a heterodimeric form of the enzyme for comparison with the homodimer. The heterodimer comprises an active wild‐type monomer and an inactive monomer in which an active‐site tyrosine is mutated to phenylalanine (Y382F). This mutation prevents the formation of the trihydroxyphenylalanine quinone (TPQ) cofactor. A pETDuet vector and a dual fusion tag strategy was used to purify heterodimers (WT/Y382F) from homodimers. Purity was confirmed by western blot and native PAGE analyses. Spectral and kinetic studies support the view that whether there are one or two functional monomers in the dimer, the properties of each functional monomer are the same, thus indicating no communication between the active sites in this bacterial enzyme.     

Ketoreduction in mycolactone biosynthesis: insight into substrate specificity and stereocontrol from studies of discrete ketoreductase domains in vitro

Mycolactone, a polyketide toxin responsible for the extensive tissue destruction seen in Buruli ulcer, is assembled on a modular polyketide synthase (PKS). Despite operating on structurally different intermediates during synthesis, many of the ketoreductase (KR) domains of the mycolactone (MLS) PKS have identical sequences. This suggests that these enzymes might exhibit an unusually high level of substrate promiscuity. However, we show here that when recombinant mycolactone KR domains are tested with a range of surrogate substrates, their specificity closely matches that of KR domains derived from other PKS systems. In addition, our findings reinforce the role of substrate tethering for achieving stereochemical control in modular PKSs by affecting the delicate energetics of ketoreduction.     

Deciphering the Binding Interactions between Acinetobacter baumannii ACP and β-ketoacyl ACP Synthase III to Improve Antibiotic Targeting Using NMR Spectroscopy

Fatty acid synthesis is essential for bacterial viability. Thus, fatty acid synthases (FASs) represent effective targets for antibiotics. Nevertheless, multidrug-resistant bacteria, including the human opportunistic bacteria, Acinetobacter baumannii, are emerging threats. Meanwhile, the FAS pathway of A. baumannii is relatively unexplored. Considering that acyl carrier protein (ACP) has an important role in the delivery of fatty acyl intermediates to other FAS enzymes, we elucidated the solution structure of A. baumannii ACP (AbACP) and, using NMR spectroscopy, investigated its interactions with β-ketoacyl ACP synthase III (AbKAS III), which initiates fatty acid elongation. The results show that AbACP comprises four helices, while Ca²⁺ reduces the electrostatic repulsion between acid residues, and the unconserved F47 plays a key role in thermal stability. Moreover, AbACP exhibits flexibility near the hydrophobic cavity entrance from D59 to T65, as well as in the α₁α₂ loop region. Further, F29 and A69 participate in slow exchanges, which may be related to shuttling of the growing acyl chain. Additionally, electrostatic interactions occur between the α₂ and α₃-helix of ACP and AbKAS III, while the hydrophobic interactions through the ACP α₂-helix are seemingly important. Our study provides insights for development of potent antibiotics capable of inhibiting A. baumannii FAS protein–protein interactions.     

离子印迹聚合物功能单体的研究进展

阐述了不饱和羧酸类（甲基丙烯酸、丙烯酸和乙烯基苯甲酸）、芳香杂环类（吡啶类、咪唑类和喹啉类）、水杨醛类希夫碱以及Salen配体等功能单体的性能、优点、用途等方面的研究进展,以及基于上述单体制备的离子印迹聚合物吸附特性和应用性能等,展望了离子印迹聚合物功能单体未来的发展方向：一是功能单体种类需要进一步丰富;二是其所具有的性能和功能等还有待于完善。其中,季铵盐化5-氯甲基水杨醛类希夫碱功能单体具有强亲水性能和多官能团特性,对其进行性能研究将会是探索此类功能单体在应用方面的热点内容之一。     

Dimerisation of the Yeast K+ Translocation Protein Trk1 Depends on the K+ Concentration

In baker’s yeast (Saccharomyces cerevisiae), Trk1, a member of the superfamily of K-transporters (SKT), is the main K⁺ uptake system under conditions when its concentration in the environment is low. Structurally, Trk1 is made up of four domains, each similar and homologous to a K-channel α subunit. Because most K-channels are proteins containing four channel-building α subunits, Trk1 could be functional as a monomer. However, related SKT proteins TrkH and KtrB were crystallised as dimers, and for Trk1, a tetrameric arrangement has been proposed based on molecular modelling. Here, based on Bimolecular Fluorescence Complementation experiments and single-molecule fluorescence microscopy combined with molecular modelling; we provide evidence that Trk1 can exist in the yeast plasma membrane as a monomer as well as a dimer. The association of monomers to dimers is regulated by the K⁺ concentration.     

Functional Characterization of PyrG,an Unusual Nonribosomal Peptide Synthetase Module from the Pyridomycin Biosynthetic Pathway

Pyridomycin is an antimycobacterial cyclodepsipeptide assembled by a nonribosomal peptide synthetase/polyketide synthase hybrid system. Analysis of its cluster revealed a nonribosomal peptide synthetase (NRPS) module, PyrG, that contains two tandem adenylation domains and a PKS‐type ketoreductase domain. In this study, we biochemically validated that the second A domain recognizes and activates α‐keto‐β‐methylvaleric acid (2‐KVC) as the native substrate; the first A domain was not functional but might play a structural role. The KR domain catalyzed the reduction of the 2‐KVC tethered to the peptidyl carrier protein of PyrG in the presence of the MbtH family protein, PyrH. PyrG was demonstrated to recognize many amino acids. This substrate promiscuity provides the potential to generate pyridomycin analogues with various enolic acids moiety; this is important for binding InhA, a critical enzyme for cell‐wall biosynthesis in Mycobacterium tuberculosis.