Information

Are some polyketides enzymes?

Are some polyketides enzymes?


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

I am currently reading a "book" (rather an article) called "Protein Modelling & Molecular Docking: Modeller, Autodock".

The abstract starts with the following sentence :

Polyketides are a large family of complex enzymes synthesized by polyketide synthases.

Before reading this article, I knew nothing about polyketides. I looked it up online, and I saw that they were secondary metabolites, which adds up with the rest of the article. However, I could not find any mention of some polyketides being enzymes, hence my question:

Are some polyketides enzymes or is this a simple mistake in the article ?


You are correct. Polyketide synthases (PKS) are the enzymes that synthesize polyketides. PKS are derived from fatty acid synthases via evolution. Polyketides are of great interest becaus many are of important medical use.


PKSs can be classified into three groups with the following subdivisions:

  • Type I polyketide synthases are large, highly modular proteins.
    • Iterative Type I PKSs reuse domains in a cyclic fashion.
      • NR-PKSs — non-reducing PKSs, the products of which are true polyketides
      • PR-PKSs — partially reducing PKSs
      • FR-PKSs — fully reducing PKSs, the products of which are fatty acid derivatives

      Each type I polyketide-synthase module consists of several domains with defined functions, separated by short spacer regions. The order of modules and domains of a complete polyketide-synthase is as follows (in the order N-terminus to C-terminus):

      • Starting or loading module: AT-ACP-
      • Elongation or extending modules: -KS-AT-[DH-ER-KR]-ACP-
      • Termination or releasing domain: -TE
      • AT: Acyltransferase
      • ACP: Acyl carrier protein with an SH group on the cofactor, a serine-attached 4'-phosphopantetheine
      • KS: Keto-synthase with an SH group on a cysteine side-chain
      • KR: Ketoreductase
      • DH: Dehydratase
      • ER: Enoylreductase
      • MT: Methyltransferase O- or C- (α or β)
      • SH: PLP-dependent cysteine lyase
      • TE: Thioesterase

      The polyketide chain and the starter groups are bound with their carboxy functional group to the SH groups of the ACP and the KS domain through a thioester linkage: R-C(=O)OH + HS-protein <=> R-C(=O)S-protein + H2O.

      The ACP carrier domains are similar to the PCP carrier domains of nonribosomal peptide synthetases, and some proteins combine both types of modules.

      The growing chain is handed over from one thiol group to the next by trans-acylations and is released at the end by hydrolysis or by cyclization (alcoholysis or aminolysis).

      • The starter group, usually acetyl-CoA or its analogues, is loaded onto the ACP domain of the starter module catalyzed by the starter module's AT domain.
      • The polyketide chain is handed over from the ACP domain of the previous module to the KS domain of the current module, catalyzed by the KS domain.
      • The elongation group, usually malonyl-CoA or methylmalonyl-CoA, is loaded onto the current ACP domain catalyzed by the current AT domain.
      • The ACP-bound elongation group reacts in a Claisen condensation with the KS-bound polyketide chain under CO2 evolution, leaving a free KS domain and an ACP-bound elongated polyketide chain. The reaction takes place at the KSn-bound end of the chain, so that the chain moves out one position and the elongation group becomes the new bound group.
      • Optionally, the fragment of the polyketide chain can be altered stepwise by additional domains. The KR (keto-reductase) domain reduces the β-keto group to a β-hydroxy group, the DH (dehydratase) domain splits off H2O, resulting in the α-β-unsaturated alkene, and the ER (enoyl-reductase) domain reduces the α-β-double-bond to a single-bond. It is important to note that these modification domains actually affect the previous addition to the chain (i.e. the group added in the previous module), not the component recruited to the ACP domain of the module containing the modification domain.
      • This cycle is repeated for each elongation module.

      Polyketide synthases are an important source of naturally occurring small molecules used for chemotherapy. [3] For example, many of the commonly used antibiotics, such as tetracycline and macrolides, are produced by polyketide synthases. Other industrially important polyketides are sirolimus (immunosuppressant), erythromycin (antibiotic), lovastatin (anticholesterol drug), and epothilone B (anticancer drug). [4]

      Only about 1% of all known molecules are natural products, yet it has been recognized that almost two thirds of all drugs currently in use are at least in part derived from a natural source. [5] This bias is commonly explained with the argument that natural products have co-evolved in the environment for long time periods and have therefore been pre-selected for active structures. Polyketide synthase products include lipids with antibiotic, antifungal, antitumor, and predator-defense properties however, many of the polyketide synthase pathways that bacteria, fungi and plants commonly use have not yet been characterized. [6] [7] Methods for the detection of novel polyketide synthase pathways in the environment have therefore been developed. Molecular evidence supports the notion that many novel polyketides remain to be discovered from bacterial sources. [8] [9]


      Primary Metabolism of Human Pathogenic Fungi, Importance for Virulence and Potential for Drug Development

      Jennifer Scott , Jorge Amich , in Reference Module in Biomedical Sciences , 2021

      Polyketides

      Polyketides are a rare type of lipids, normally referred as secondary metabolites, that have a biosynthetic chemistry highly similar to fatty acids ( Smith and Tsai, 2007 Chiang et al., 2010 ). This diverse group of compounds has great significance for the virulence of fungal pathogens. Particularly, Aspergillus species can produce many polyketides, by action of polyketide synthase enzymes type I, with high relevance to human health ( Bhetariya et al., 2011 ). For example, A. flavus and A. parasiticus produce aflatoxin, by a polyketide pathway consisting of at least 27 reactions, which is a toxin that contaminates food and causes hepatotoxicity, teratogenicity, and immunotoxicity ( Caceres et al., 2020 Kumar et al., 2017 ). Furthermore, A. fumigatus produces (among approximately 8 polyketides) DHN-melanin, which covers spores and that is known to be a very important fungal virulence factor ( Bignell et al., 2016 Heinekamp et al., 2013 Akoumianaki et al., 2016 ).


      Are some polyketides enzymes? - Biology

      Polyketides are a class of natural products isolated from microbes, plants and invertebrates which includes an impressive number of clinically effective drugs with diverse activities. To name a few examples: erythromycin (antibiotic), rapamycin (immunosuppressive), amphotericin (antifungal), avermectin (antiparasitic), and doxorubicin (anticancer). As other natural products do, polyketides may play disparate roles in the producing organisms, from defensive weapons (inhibiting growth of competitors, or acting against predators) to signaling molecules (working as messengers between social organisms). In Mycobacterium tuberculosis , some polyketides are key intermediates in the synthesis of complex lipids. These lipids are important components of the unusually thick cell envelope, and help the microbe to be a successful pathogen. Therefore, the study of polyketide synthesis in this bacterium may lead to the design of specific inhibitors as new anti-mycobacterial drugs.

      Polyketides are produced through a stepwise condensation of simple carboxylic acid precursors, resembling fatty acid biosythesis. This task is performed by enzymes known as polyketide synthases (PKSs). There are several types of PKSs, from relatively simple proteins to large multienzymatic complexes possessing tens of catalytic sites. They use any of two general mechanisms: (1) modular — in which each set of catalytic sites is used only once during the biosynthetic process, and (2) iterative — in which the same set of active sites is used repeatedly. This week in PLoS Biology , Rajesh Gokhale and colleagues present their research involving a peculiar PKS from M. tuberculosis . The PKS12 protein is encoded by the largest gene in the microbe's genome, and participates in the synthesis of an antigenic phosphoglycolipid. Most remarkably, this PKS appears to use a new hybrid "modularly iterative" mechanism for polyketide synthesis. Several molecules of the PKS12 protein join together to form a supramolecular assembly, which performs repetitive cycles of iterations. The protein assembly is formed by specific intermolecular interactions between N- and C-terminus linkers. This study provides another example of the catalytic and mechanistic versatility of PKSs — natural product biosynthesis is an inexhaustible source for new biochemistry!

      Citation (open access):
      Chopra T, Banerjee S, Gupta S, Yadav G, Anand S, Surolia A, Roy RP, Mohanty D, Gokhale RS (2008). Novel intermolecular iterative mechanism for biosynthesis of mycoketide catalyzed by a bimodular polyketide synthase. PLoS Biology 6(7), e163. DOI: 10.1371/journal.pbio.0060163

      Image: model of the PKS12 protein, modified from Figure 5 of the cited article.

        . PLoS Biology (2004) 2(2): e35. (video of an excellent talk by Chaitan Khosla, May 2007). iBioSeminars, American Society for Cell Biology. The first part of the talk (Polyketides and Polyketide Biosynthesis) is also available at Google Video, and is embedded below.

      Polyketides are a class of natural products isolated from microbes, plants and invertebrates which includes an impressive number of clinically effective drugs with diverse activities. To name a few examples: erythromycin (antibiotic), rapamycin (immunosuppressive), amphotericin (antifungal), avermectin (antiparasitic), and doxorubicin (anticancer). As other natural products do, polyketides may play disparate roles in the producing organisms, from defensive weapons (inhibiting growth of competitors, or acting against predators) to signaling molecules (working as messengers between social organisms). In Mycobacterium tuberculosis , some polyketides are key intermediates in the synthesis of complex lipids. These lipids are important components of the unusually thick cell envelope, and help the microbe to be a successful pathogen. Therefore, the study of polyketide synthesis in this bacterium may lead to the design of specific inhibitors as new anti-mycobacterial drugs.

      Polyketides are produced through a stepwise condensation of simple carboxylic acid precursors, resembling fatty acid biosythesis. This task is performed by enzymes known as polyketide synthases (PKSs). There are several types of PKSs, from relatively simple proteins to large multienzymatic complexes possessing tens of catalytic sites. They use any of two general mechanisms: (1) modular — in which each set of catalytic sites is used only once during the biosynthetic process, and (2) iterative — in which the same set of active sites is used repeatedly. This week in PLoS Biology , Rajesh Gokhale and colleagues present their research involving a peculiar PKS from M. tuberculosis . The PKS12 protein is encoded by the largest gene in the microbe's genome, and participates in the synthesis of an antigenic phosphoglycolipid. Most remarkably, this PKS appears to use a new hybrid "modularly iterative" mechanism for polyketide synthesis. Several molecules of the PKS12 protein join together to form a supramolecular assembly, which performs repetitive cycles of iterations. The protein assembly is formed by specific intermolecular interactions between N- and C-terminus linkers. This study provides another example of the catalytic and mechanistic versatility of PKSs — natural product biosynthesis is an inexhaustible source for new biochemistry!

      Citation (open access):
      Chopra T, Banerjee S, Gupta S, Yadav G, Anand S, Surolia A, Roy RP, Mohanty D, Gokhale RS (2008). Novel intermolecular iterative mechanism for biosynthesis of mycoketide catalyzed by a bimodular polyketide synthase. PLoS Biology 6(7), e163. DOI: 10.1371/journal.pbio.0060163

      Image: model of the PKS12 protein, modified from Figure 5 of the cited article.

        . PLoS Biology (2004) 2(2): e35. (video of an excellent talk by Chaitan Khosla, May 2007). iBioSeminars, American Society for Cell Biology. The first part of the talk (Polyketides and Polyketide Biosynthesis) is also available at Google Video, and is embedded below.


      Except where otherwise noted, blog posts by Cesar Sanchez in Twisted Bacteria are licensed under a Creative Commons Attribution 3.0 Unported License. Please let me know if any quotes or images on this blog are improperly credited. E-mail: TwistedBacteria AT gmail DOT com . Social media icons by Oliver Twardowski and AddThis.


      Lipid metabolism

      Very few organisms produce such a diverse array of lipophilic molecules as M. tuberculosis. These molecules range from simple fatty acids such as palmitate and tuberculostearate, through isoprenoids, to very-long-chain, highly complex molecules such as mycolic acids and the phenolphthiocerol alcohols that esterify with mycocerosic acid to form the scaffold for attachment of the mycosides. Mycobacteria contain examples of every known lipid and polyketide biosynthetic system, including enzymes usually found in mammals and plants as well as the common bacterial systems. The biosynthetic capacity is overshadowed by the even more remarkable radiation of degradative, fatty acid oxidation systems and, in total, there are ∼ 250 distinct enzymes involved in fatty acid metabolism in M. tuberculosis compared with only 50 in E. coli 20 .

      Fatty acid degradation. In vivo-grown mycobacteria have been suggested to be largely lipolytic, rather than lipogenic, because of the variety and quantity of lipids available within mammalian cells and the tubercle 2 (Fig. 4a). The abundance of genes encoding components of fatty acid oxidation systems found by our genomic approach supports this proposition, as there are 36 acyl-CoA synthases and a family of 36 related enzymes that could catalyse the first step in fatty acid degradation. There are 21 homologous enzymes belonging to the enoyl-CoA hydratase/isomerase superfamily of enzymes, which rehydrate the nascent product of the acyl-CoA dehydrogenase. The four enzymes that convert the 3-hydroxy fatty acid into a 3-keto fatty acid appear less numerous, mainly because they are difficult to distinguish from other members of the short-chain alcohol dehydrogenase family on the basis of primary sequence. The five enzymes that complete the cycle by thiolysis of the β-ketoester, the acetyl-CoA C-acetyltransferases, do indeed appear to be a more limited family. In addition to this extensive set of dissociated degradative enzymes, the genome also encodes the canonical FadA/FadB β-oxidation complex (Rv0859 and Rv0860). Accessory activities are present for the metabolism of odd-chain and multiply unsaturated fatty acids.

      a, Degradation of host-cell lipids is vital in the intracellular life of M. tuberculosis. Host-cell membranes provide precursors for many metabolic processes, as well as potential precursors of mycobacterial cell-wall constituents, through the actions of a broad family of β-oxidative enzymes encoded by multiple copies in the genome. These enzymes produce acetyl CoA, which can be converted into many different metabolites and fuel for the bacteria through the actions of the enzymes of the citric acid cycle and the glyoxylate shunt of this cycle. b, The genes that synthesize mycolic acids, the dominant lipid component of the mycobacterial cell wall, include the type I fatty acid synthase (fas) and a unique type II system which relies on extension of a precursor bound to an acyl carrier protein to form full-length ( ∼ 80-carbon) mycolic acids. The cma genes are responsible for cyclopropanation. c, The genes that produce phthiocerol dimycocerosate form a large operon and represent type I (mas) and type II (the pps operon) polyketide synthase systems. Functions are colour coordinated.

      Fatty acid biosynthesis. At least two discrete types of enzyme system, fatty acid synthase (FAS) I and FAS II, are involved in fatty acid biosynthesis in mycobacteria (Fig. 4b). FAS I (Rv2524, fas) is a single polypeptide with multiple catalytic activities that generates several shorter CoA esters from acetyl-CoA primers 5 and probably creates precursors for elongation by all of the other fatty acid and polyketide systems. FAS II consists of dissociable enzyme components which act on a substrate bound to an acyl-carrier protein (ACP). FAS II is incapable of de novo fatty acid synthesis but instead elongates palmitoyl-ACP to fatty acids ranging from 24 to 56 carbons in length 17 , 21 . Several different components of FAS II may be targets for the important tuberculosis drug isoniazid, including the enoyl-ACP reductase InhA 22 , the ketoacyl-ACP synthase KasA and the ACP AcpM 21 . Analysis of the genome shows that there are only three potential ketoacyl synthases: KasA and KasB are highly related, and their genes cluster with acpM, whereas KasC is a more distant homologue of a ketoacyl synthase III system. The number of ketoacyl synthase and ACP genes indicates that there is a single FAS II system. Its genetic organization, with two clustered ketoacyl synthases, resembles that of type II aromatic polyketide biosynthetic gene clusters, such as those for actinorhodin, tetracycline and tetracenomycin in Streptomyces species 23 . InhA seems to be the sole enoyl-ACP reductase and its gene is co-transcribed with a fabG homologue, which encodes 3-oxoacyl-ACP reductase. Both of these proteins are probably important in the biosynthesis of mycolic acids.

      Fatty acids are synthesized from malonyl-CoA and precursors are generated by the enzymatic carboxylation of acetyl (or propionyl)-CoA by a biotin-dependent carboxylase (Fig. 4b). From study of the genome we predict that there are three complete carboxylase systems, each consisting of an α- and a β-subunit, as well as three β-subunits without an α-counterpart. As a group, all of the carboxylases seem to be more related to the mammalian homologues than to the corresponding bacterial enzymes. Two of these carboxylase systems (accA1, accD1 and accA2, accD2) are probably involved in degradation of odd-numbered fatty acids, as they are adjacent to genes for other known degradative enzymes. They may convert propionyl-CoA to succinyl-CoA, which can then be incorporated into the tricarboxylic acid cycle. The synthetic carboxylases (accA3, accD3, accD4, accD5 and accD6) are more difficult to understand. The three extra β-subunits might direct carboxylation to the appropriate precursor or may simply increase the total amount of carboxylated precursor available if this step were rate-limiting.

      Synthesis of the paraffinic backbone of fatty and mycolic acids in the cell is followed by extensive postsynthetic modifications and unsaturations, particularly in the case of the mycolic acids 24 , 25 . Unsaturation is catalysed either by a FabA-like β-hydroxyacyl-ACP dehydrase, acting with a specific ketoacyl synthase, or by an aerobic terminal mixed function desaturase that uses both molecular oxygen and NADPH. Inspection of the genome revealed no obvious candidates for the FabA-like activity. However, three potential aerobic desaturases (encoded by desA1, desA2 and desA3) were evident that show little similarity to related vertebrate or yeast enzymes (which act on CoA esters) but instead resemble plant desaturases (which use ACP esters). Consequently, the genomic data indicate that unsaturation of the meromycolate chain may occur while the acyl group is bound to AcpM.

      Much of the subsequent structural diversity in mycolic acids is generated by a family of S-adenosyl- L -methionine-dependent enzymes, which use the unsaturated meromycolic acid as a substrate to generate cis and trans cyclopropanes and other mycolates. Six members of this family have been identified and characterized 25 and two clustered, convergently transcribed new genes are evident in the genome ( umaA1 and umaA2). From the functions of the known family members and the structures of mycolic acids in M. tuberculosis, it is tempting to speculate that these new enzymes may introduce the trans cyclopropanes into the meromycolate precursor. In addition to these two methyltransferases, there are two other unrelated lipid methyltransferases (Ufa1 and Ufa2) that share homology with cyclopropane fatty acid synthase of E. coli 25 . Although cyclopropanation seems to be a relatively common modification of mycolic acids, cyclopropanation of plasma-membrane constituents has not been described in mycobacteria. Tuberculostearic acid is produced by methylation of oleic acid, and may be synthesized by one of these two enzymes.

      Condensation of the fully functionalized and preformed meromycolate chain with a 26-carbon α-branch generates full-length mycolic acids that must be transported to their final location for attachment to the cell-wall arabinogalactan. The transfer and subsequent transesterification is mediated by three well-known immunogenic proteins of the antigen 85 complex 26 . The genome encodes a fourth member of this complex, antigen 85C′ (fbpC2, Rv0129), which is highly related to antigen 85C. Further studies are needed to show whether the protein possesses mycolytransferase activity and to clarify the reason behind the apparent redundancy.

      Polyketide synthesis. Mycobacteria synthesize polyketides by several different mechanisms. A modular type I system, similar to that involved in erythromycin biosynthesis 23 , is encoded by a very large operon, ppsABCDE, and functions in the production of phenolphthiocerol 5 . The absence of a second type I polyketide synthase suggests that the related lipids phthiocerol A and B, phthiodiolone A and phthiotriol may all be synthesized by the same system, either from alternative primers or by differential postsynthetic modification. It is physiologically significant that the pps gene cluster occurs immediately upstream of mas, which encodes the multifunctional enzyme mycocerosic acid synthase (MAS), as their products phthiocerol and mycocerosic acid esterify to form the very abundant cell-wall-associated molecule phthiocerol dimycocerosate (Fig. 4c).

      Members of another large group of polyketide synthase enzymes are similar to MAS, which also generates the multiply methyl-branched fatty acid components of mycosides and phthiocerol dimycocerosate, abundant cell-wall-associated molecules 5 . Although some of these polyketide synthases may extend type I FAS CoA primers to produce other long-chain methyl-branched fatty acids such as mycolipenic, mycolipodienic and mycolipanolic acids or the phthioceranic and hydroxyphthioceranic acids, or may even show functional overlap 5 , there are many more of these enzymes than there are known metabolites. Thus there may be new lipid and polyketide metabolites that are expressed only under certain conditions, such as during infection and disease.

      A fourth class of polyketide synthases is related to the plant enzyme superfamily that includes chalcone and stilbene synthase 23 . These polyketide synthases are phylogenetically divergent from all other polyketide and fatty acid synthases and generate unreduced polyketides that are typically associated with anthocyanin pigments and flavonoids. The function of these systems, which are often linked to apparent type I modules, is unknown. An example is the gene cluster spanning pks10, pks7, pks8 and pks9, which includes two of the chalcone-synthase-like enzymes and two modules of an apparent type I system. The unknown metabolites produced by these enzymes are interesting because of the potent biological activities of some polyketides such as the immunosuppressor rapamycin.

      Siderophores. Peptides that are not ribosomally synthesized are made by a process that is mechanistically analogous to polyketide synthesis 23 , 27 . These peptides include the structurally related iron-scavenging siderophores, the mycobactins and the exochelins 2 , 28 , which are derived from salicylate by the addition of serine (or threonine), two lysines and various fatty acids and possible polyketide segments. The mbt operon, encoding one apparent salicylate-activating protein, three amino-acid ligases, and a single module of a type I polyketide synthase, may be responsible for the biosynthesis of the mycobacterial siderophores. The presence of only one non-ribosomal peptide-synthesis system indicates that this pathway may generate both siderophores and that subsequent modification of a single ε-amino group of one lysine residue may account for the different physical properties and function of the siderophores 28 .


      Multidrug- and Pandrug-Resistant Bacteria

      It has been long known that microorganisms have several mechanisms of drug resistance, which arise from natural selection of the “gene pool” of constantly evolving microbial populations either by gene mutations (vertical evolution) or by the acquisition of genes from other non-related species through mobile genetic elements such as plasmids, phages and transposons (i.e., horizontal gene transfer). Figure 3A shows some of the major mechanisms of bacterial resistance to known antibiotics. Historically, the inappropriate use of pharmacological compounds in the clinical setting has placed artificial selection on the microbiota, accelerating the spread of resistance among pathogen and commensal microorganisms. As observed in Figure 3B , the emergence of resistant pathogens in a hospital environment typically occurs shortly after the introduction of a new compound (Schmieder and Edwards, 2012).

      Mechanisms of bacterial resistance to antibiotics (A) and Antibiotics resistance evolution showing the rapid development of resistance to multiple classes of antibiotics (B). Reproduced from Future Microbiology, January 2012, Vol. 7, No. 1, Pages 73� with permission of Future Medicine Ltd. (Schmieder and Edwards, 2012).

      Despite this situation, the emergence of genes related to antibiotic resistance preceded the clinical use of antibiotics by humans and should not be understood as the result of this selection but as its raw material. Microbial antibiotic resistance and pathogenicity are not modern phenomena. Studies using metagenomic DNA samples extracted from Beringian permafrost sediments that were protected from contamination by layers of volcanic tephra (C 14 dated at 30,000 years old, belonging to the Pleistocene) revealed the presence of several gene clusters encoding resistance to β-lactam, tetracyclines, glycopeptide antibiotics and the presence of sequence variants similar to the modern vancomycin resistance element VanA (D𠆜osta et al., 2011). This demonstrates the natural course of the emergence of these genes through evolution, suggesting that their role in the antagonistic relations within the microbial community precedes the effects of human action.

      If the constant adaptation of microorganisms to drugs in the circulation has required a constant search for new formulations, then the emergence of multidrug resistant bacteria classes has made the discovery of novel chemical molecules a priority. Some promising approaches have emerged, such as prospecting in unexplored bacterial niches, database searching for synthetic molecules (Fischbach and Walsh, 2009) and the use of metagenomics.

      The first class corresponds to the methicillin-resistant Staphylococcus aureus (MRSA), and this group is identified as the cause of 19,000 annual deaths in the United States and an annual increase in health costs on the order of 3 to 4 billion dollars. As an aggravating factor, the prevalence of MRSA increases the likelihood of vancomycin resistant S. aureus (VRSA), further challenging disease treatment in hospitals (Fischbach and Walsh, 2009).

      The second class includes gram-positive multidrug-resistant (MDR with resistance to certain drugs) and pandrug-resistant (PDR with resistance to all drugs) bacteria, which are less prevalent than MRSA, which are albeit related to incurable clinical pictures. The strains Acinetobacter baumannii, Escherichia coli, Klebsiella pneunominae, and Pseudomonas aeruginosa are MDR/PDR to penicillin, cephalosporin, carbapenem, monobactam, quinolone, aminoglycoside, tetracycline and polymyxin (Fischbach and Walsh, 2009).

      The third class is comprised of Mycobacterium tuberculosis strains that are MDR and extensively drug resistant (XDR), and they are a growing problem in developing countries, requiring treatment for two years with antibiotics that cause severe side effects (Fischbach and Walsh, 2009).

      This situation brings up the discussion of the misuse of drugs, which for years has accelerated this resistance process that is caused by generations of microbes that are resurgent from treatments that are insufficient in duration and dose. This concern has also led to the creation of stricter rules for the purchase of antibiotics such as the recent decision by the Brazilian National Health Surveillance Agency (Agência Nacional de Vigilância Sanitária - ANVISA) for the mandatory submission of a prescription to purchase antibiotics, thereby curbing self-medication.


      II. Protein–protein interaction between AT and ACP in vicenistatin biosynthesis

      ATs are responsible for the selection and incorporation of acyl starter and extender units in polyketide biosynthesis. 22 , ) Therefore, ATs could be attractive targets to alter the specificity of the acyl building block to obtain biologically active unnatural polyketide analogs. However, the replacement of an AT domain by a homologous AT domain possessing different acyl substrate specificity resulted in reduced or abolished production of polyketide analogs in many cases. 22,23 ) AT was reported to recognize its cognate ACP from other ACPs through a protein–protein interaction. 24,25 ) Thus, a proper protein–protein interaction between AT and ACP is important for functional transfer of acyl groups in polyketide biosynthesis. However, the mechanism of ACP recognition is not well understood because structural determination of the AT–ACP complex is hampered by the weak and transient interaction between them. 26 ) Structural determination of the AT–ACP complex is necessary for understanding the ACP recognition mechanism during the acyl transfer reaction.

      In vicenistatin biosynthesis, VinK is supposed to recognize VinL and VinP1LdACP from other ACPs for the transfer of the dipeptidyl group. To visualize the specific protein–protein interaction between VinK and two ACPs, we attempted to cocrystallize VinK with VinL and VinP1LdACP. To trap the transient VinK–ACP complexes, a covalent cross-linking method using a bifunctional maleimide reagent was designed. 14 , ) The complex structure of P450cam with the redox partner putidaredoxin was determined previously by trapping the transient complex using 1,6-bismaleimidohexane. 27 , ) In this case, surface residues of these proteins were mutated to Cys, enabling site-specific cross-linking to occur at their binding interface. In VinK–ACP, a cross-link with the thiol group of the phosphopantetheine arm of ACP at the substrate-binding tunnel of VinK was proposed (Fig. 3(F)). For this purpose, a Cys mutation at Ser266 was introduced, which is located at the bottom of the substrate-binding tunnel in VinK. The cross-linking reaction between the VinK S266C mutant and VinL in the presence of 1,2-bismaleimidoethane (BMOE) gave a covalent complex, as expected. Finally, the VinK–VinL complex structure was determined successfully, which is the first crystal structure of an AT–ACP complex. 14 ) Similarly, the cross-linking reaction between the VinK S266C mutant and VinP1LdACP in the presence of BMOE gave a covalent complex however, obtaining a crystal of VinK–VinP1LdACP complex failed.

      In the VinK–VinL complex structure, the phosphopantetheine arm of VinL is orientated into the VinK substrate-binding pocket and covalently attached to the mutated Cys266 of VinK through BMOE (Fig. 3(G)). The binding interface between VinK and VinL comprises approximately 650 Å 2 . This small contact area is consistent with the transient nature of the AT–ACP interaction. VinK mainly recognizes the helix II region of VinL through salt bridges and hydrophobic interactions (Fig. 3(G)). Arg153 and Arg299 of VinK form salt bridges with Glu47 and Asp35 of VinL, respectively. Met206 of VinK forms hydrophobic contacts with Thr39, Leu43, Leu59, and Phe64 of VinL. VinK R153A, M206A, and R299A mutants showed significantly reduced affinities for VinL, confirming the importance of these VinK residues for the interaction with VinL. Based on the VinK–VinL complex structure, insight into the recognition mechanism of VinP1LdACP by VinK was also obtained. The VinK–VinL complex structure could be useful as a model for predicting the interaction of other AT–ACP complexes.

      Most ATs accept acyl-CoA as a substrate for transfer of the acyl group to the partner ACP. These acyl-CoA-specific ATs generally have an Arg or Lys residue near the entrance of the substrate-binding tunnel for interaction with the phosphate group of the ribose moiety of CoA. 15 , ) In contrast, VinK has a hydrophobic Met206 residue, which interacts with VinL residues, at the corresponding position. Other acyl-ACP-specific ATs such as ZmaA, 28 , ) ZmaF, 28 , ) and ClbG 29 ) also contain a hydrophobic residue at this position. Thus, the presence of a hydrophobic residue at this position might be a conserved feature in acyl-ACP-specific ATs.


      BIOSYNTHESIS OF NOVEL MOLECULES BY TYPE III PKSs THROUGH PRECURSOR-DIRECTED AND MUTAGENESIS APPROACHES

      Type III PKSs have been used for the synthesis of novel molecules because of their broad substrate specificity. Notably, incorporation of unnatural starter units has shown significance in generating new compounds. The BAS from R. palmatum catalyzes a simple decarboxylative condensation of malonyl-CoA with 4-coumaroyl-CoA. Taking advantage of its unusually broad substrate specificity and notable catalytic versatility, Wakimoto et al. synthesized a series of unnatural, novel tetramic acid derivatives by using aminoacyl-CoA thioesters as the starter units (Fig. 9). One of the tetramic acid products formed from D -phenylalanoyl-CoA showed moderate antiproliferative activity against murine leukemia P388 cells ( 46 ).

      Biosynthesis of novel cytotoxic tetramic acid derivatives by BAS.

      The structure-based mutagenesis of type III PKSs has been demonstrated to be a useful approach to engineering these enzymes and expanding the catalytic repertoire to produce larger and more complex polyketide molecules. As described above, the wild-type OKS from A. arborescens catalyzes the condensation of eight molecules of malonyl-CoA to produce the aromatic octaketides SEK4 and SEK4b (Fig. 2). The enzyme was first modified by a single mutation to generate the N222G mutant, which was able to produce a longer decaketide benzophenone SEK15 (Fig. 10A). Furthermore, the F66L/N222G double mutant was shown to produce dodecaketide TW95a by sequential condensation of 12 molecules of malonyl-CoA (Fig. 10B). A homology model predicted that the volume of the active-site cavity in the F66L/N222G mutant is increased to 748 Å, from 652 Å of the wild-type OKS ( 47 ), which explains why the longer polyketide can be synthesized.

      Engineered biosynthesis of long polyketides by OAS. (A) Biosynthesis of SEK15 by the N222G mutant of OAS. (B) Biosynthesis of TW95a by the F66L/N222G mutant of OAS.

      A recent work on HsPKS1 from Huperzia serrata has demonstrated that engineering of type III PKSs will not only lead to the synthesis of regular polyketides but also generate other types of molecules. HsPKS1 is similar to CHS, catalyzing the sequential condensation of 4-coumaroyl-CoA with three units of malonyl-CoA. However, HsPKS1 has an unusually broad substrate specificity, which allows the biosynthesis of unnatural unique polyketide-alkaloid hybrid molecules. This occurs via the condensation of nitrogen-containing substrates with two molecules of malonyl-CoA. The structure-based S348G mutant extended the product chain length and altered the cyclization mechanism. This is exemplified by the reactions in Fig. 11. HsPKS1 catalyzes the condensation of 2-carbamoylbenzoyl-CoA with two units of malonyl-CoA to generate 2-hydroxypyrido[2,1-a]isoindole-4,6-dione (Fig. 11A), whereas the S348G mutant takes three units of the extender units, yielding a biologically active alkaloid, 1,3-dihydroxy-5H-dibenzo[b,e]azepine-6,11-dioine (Fig. 11B) ( 48 ). An earlier research also reported the biosynthesis of 4-hydroxy-2(1H)-quinolones, a novel alkaloidal scaffold, by the BAS from R. palmatum with N-methylanthraniloyl-CoA and anthraniloyl-CoA as the starter units ( 49 ). These results have revealed that the use of nonphysiological substrates by type III PKSs may yield novel molecules.

      Biosynthesis of novel alkaloids by HsPKS1. (A) Biosynthesis of 2-hydroxypyrido[2,1-a]isoindole-4,6-dione by HsPKS1 from a unnatural starter unit, 2-carbamoylbenzoyl-CoA. (B) Engineered biosynthesis of 1,3-dihydroxy-5H-dibenzo[b,e]azepine-6,11-dioine by the S348G mutant of HsPKS1.

      Various type III PKSs have been engineered into E. coli to generate novel polyketides. The production of plant-specific curcuminoids has been reconstituted in E. coli by coexpressing CUS with phenylalanine ammonia-lyase from Rhodotorula rubra and 4-coumarate:CoA ligase (4CL) from Lithospermum erythrorhizon ( 50 ). Furthermore, the engineered strain was utilized to produce a series of unnatural curcuminoids by providing a variety of carboxylate precursors (Fig. 12) ( 51 ). Because of its broad substrate specificity, CUS has also recently been coexpressed with a 4CL from L. erythrorhizon, a fatty acid CoA ligase from O. sativa, and a β-oxidation system from S. cerevisiae to produce gingerol derivatives (Fig. 13) ( 52 ).


      Modularity of scaffold biosynthesis

      Initiation of biosynthesis

      The composition of the polyketide backbone, or scaffold, structure is governed by the stringency of acetyltransferase domains to load a specific acyl-CoA substrate, but also through substrate stereochemistry and redox pattern ( Sundermann et al., 2013): each PKS assembles an individual product through the choice of acyl-CoA units, their level of reduction and subsequent tailoring. Initiation of scaffold biosynthesis requires selection and recruitment of a starter unit onto a didomain, comprising an acetyltransferase and an ACP, collectively termed the loading module. The resulting initial starter unit serves as the first substrate in the growth of the final β-keto-acyl chain. Generation of diversity through the promiscuity of acetyltransferase domains to load multiple different starter units, termed polyspecificity, is more commonly observed than by polyspecificity of extender modules later in biosynthesis ( McDaniel et al., 1999 Yuzawa et al., 2012). Introduction of diversity during initiation of biosynthesis also commonly occurs through the multiple different priming mechanisms used by the array of loading modules available ( Moore & Hertweck, 2002 Hertweck, 2009). Due to the mechanistic promiscuity of the starter domains, combinatorial biosynthesis attempts to manipulate PKS modules often start with here. For example, the acetyltransferase and ACP loading module of DEBS1 naturally recruit a propionate starter unit. Substitution with loading modules from tylosin and oleandomycin type I megasynthases from Streptomyces fradiae and Streptomyces antibioticus, respectively, resulted in controlled integration of propionate or acetate as a starter unit ( Long et al., 2002). Similarly, the replacement of the isobutyryl-CoA-specific loading module initiating avermectin biosynthesis in Streptomyces avermitilis M1 by the unique phoslactomycin polyketide cyclohexanecarboxylic unit loading module from Streptomyces platensis resulted in production of the veterinary antiparasitic doramectin ( Wang et al., 2011). Alteration of loading modules for the initiation of biosynthesis is therefore one step showing promise for the generation of novel polyketides.

      Chain extension

      After initiation, continued assembly of the polyketide scaffold requires loading of extender units onto the acetyltransferase and ACP and incorporation into the β-keto-acyl intermediate by the ketosynthase. At this stage, diversity can be introduced through the installation of noncanonical extender units resulting from the polyspecificity of loading domains, domain substitutions or by the iterative action of an otherwise modular PKS ( Kapur et al., 2012). The collection of commonly used extender units nature provides is modest: Canonical extender units comprise malonyl- and methylmalonyl-CoA. Substitution for domains loading other, less commonly used, extender units will allow introduction of a broadened chemistry into the polyketide backbone. For example, reductive carboxylation of α,β-unsaturated acyl-CoA precursors via crotonyl-CoA reductase/carboxylase homologues facilitates inclusion of hexyl-, propyl-, chloroethyl- and isobutylmalonyl-CoA into the polyketide scaffold ( Eustaquio et al., 2009 Liu et al., 2009 Wilson et al., 2011). Alternatively, functionalization of extender units on stand-alone ACPs allows the incorporation of allyl- ( Mo et al., 2011), amino-, hydroxyl- ( Chan et al., 2006) and methoxymalonyl-ACP ( Wu et al., 2000) extender units into the polyketide scaffold.

      Predicting the polyspecificity of extender modules to introduce these rarer extender units is not straightforward. The mechanical processing and discrimination between acyl-CoA extender units by loading domains in type I modular PKSs is currently little understood. Investigations to elucidate why particular substrates are preferred or chosen are presenting a growing body of evidence suggesting that PKSs may be able to tolerate and incorporate exogenous natural and non-natural extender units into the β-keto-acyl chain. For example, analysis of the acyl-CoA substrate selectivity of PikAIV, a pikromycin synthase from Streptomyces venezuelae, elucidated the polyspecificity of extender modules towards substrates not readily present in the producer. PikAIV successfully loaded malonyl-, propionyl-, ethyl- and native methylmalonyl-CoA to the ACP. In the case of malonyl- and propionyl-CoA, active site occupancy was low at 3% and 19%, respectively. More interestingly, the rare extender ethylmalonyl-CoA showed acetyltransferase loading of 90% and low levels of hydrolytic release indicating its potential for incorporation during assembly the native substrate methylmalonyl-CoA showed 100% acetyltransferase saturation ( Bonnett et al., 2011). In the case of PikAIV, all acyl-CoA substrates were loaded however, incorporation into the carbon chain depended upon the rate of subsequent hydrolytic release. These findings suggest that extender modules may show a greater tolerance to incorporate exogenous precursors lacking evolved selectivity, consistent with findings previously reported ( Pohl et al., 2001). Substituting extender domains for the addition of novel extender units showing limited hydrolytic release could therefore result in the generation of novel polyketides however, no concrete rules defining what properties extender modules require to do so have been elucidated, despite observed discrimination between sizes of extender units and incorporation.

      Product release

      Manipulating starter and extender modules of PKSs may permit the introduction of novel acyl-CoA substrates into the polyketide scaffold. However, for these to show activity, they must be released from the PKS. For successful release, it is important to identify which catalytic domains act as decision gates, thereby permitting continuation of downstream biosynthesis of altered β-keto-acyl intermediates. Elucidating such points will significantly aid success when engineering BGCs. Yeh et al. (2013) experimentally indicated that the phylogeny between nonreducing iterative PKS (nrPKS) modules is a good predictor of successful polyketide assembly and release from engineered BGCs. Increased phylogenetic proximity between gene units translated to improved domain–domain interactions and as a result, improved the release of the polyketide end product. In contrast, Xu et al. (2013) show for type II nrPKSs that the best predictors of thioesterase acceptance, and therefore release, are the shape and size of the polyketide substrate and consequently indicate the stringency of thioesterase domains in carrying out discriminative decision gate functions. For example, if the native substrate of a thioesterase was a nonaketide, but the engineered assembly line presented it with a heptaketide, the rate of release was almost zero ( Vagstad et al., 2013). Substituting thioesterase domains often resulted in abolished product formation, despite the presence of an abundance of β-keto-acyl intermediates produced by the upstream domains, whereas judicious choices of thioesterase substitution resulted in the successful production of an unnatural polyketide product, radilarin ( Xu et al., 2013). Successful polyketide release from thioesterase in the case of resorcyclic acid lactones and dihydroxyphenylacetate acid lactones may be dependent upon substrate size ( Xu et al., 2013). However, contrastingly, truncation of the DEBS1–3 megasynthase through relocation of the thioesterase domains downstream of the modular DEBS1 resulted in assembly and successful release of a much shortened triketide lactone ( Kao et al., 1995 Pfeifer et al., 2001). These contrasting results indicated the complex nature of the thioesterase and show the requirement for further work to build rules to predict thioesterase domain tolerance for substrates. Currently, for successful incorporation of novel starter and extender units, and successful product release, analysis of domains must be carried out on a case-by-case basis.


      Allergens/Antigens, toxins and polyketides of important Aspergillus species

      The medical, agricultural and biotechnological importance of the primitive eukaryotic microorganisms, the Fungi was recognized way back in 1920. Among various groups of fungi, the Aspergillus species are studied in great detail using advances in genomics and proteomics to unravel biological and molecular mechanisms in these fungi. Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus parasiticus, Aspergillus nidulans and Aspergillus terreus are some of the important species relevant to human, agricultural and biotechnological applications. The potential of Aspergillus species to produce highly diversified complex biomolecules such as multifunctional proteins (allergens, antigens, enzymes) and polyketides is fascinating and demands greater insight into the understanding of these fungal species for application to human health. Recently a regulator gene for secondary metabolites, LaeA has been identified. Gene mining based on LaeA has facilitated new metabolites with antimicrobial activity such as emericellamides and antitumor activity such as terrequinone A from A. nidulans. Immunoproteomic approach was reported for identification of few novel allergens for A. fumigatus. In this context, the review is focused on recent developments in allergens, antigens, structural and functional diversity of the polyketide synthases that produce polyketides of pharmaceutical and biological importance. Possible antifungal drug targets for development of effective antifungal drugs and new strategies for development of molecular diagnostics are considered.

      Keywords: Allergens Aspergillus species Polyketides.

      Figures

      Antifungal drugs and the mechanism…

      Antifungal drugs and the mechanism of action

      Lovastatin nonaketide synthase(LovB) of Aspergilllus…

      Lovastatin nonaketide synthase(LovB) of Aspergilllus terreus showing general Aspergillus Polyketide synthase domain Architect…