Architecture of full-length type I modular polyketide synthases revealed by X-ray crystallography, cryo-electron microscopy, and AlphaFold2.

Covering: up to the end of 2023Type I modular polyketide synthases construct polyketide natural products in an assembly line-like fashion, where the growing polyketide chain attached to an acyl carrier protein is passed from catalytic domain to catalytic domain. These enzymes have immense potential in drug development since they can be engineered to produce non-natural polyketides by strategically adding, exchanging, and deleting individual catalytic domains. In practice, however, this approach frequently results in complete failures or dramatically reduced product yields. A comprehensive understanding of modular polyketide synthase architecture is expected to resolve these issues. We summarize the three-dimensional structures and the proposed mechanisms of three full-length modular polyketide synthases, Lsd14, DEBS module 1, and PikAIII. We also describe the advantages and limitations of using X-ray crystallography, cryo-electron microscopy, and AlphaFold2 to study intact type I polyketide synthases.

Triepoxide formation by a flavin-dependent monooxygenase in monensin biosynthesis.

Monensin A is a prototypical natural polyether polyketide antibiotic. It acts by binding a metal cation and facilitating its transport across the cell membrane. Biosynthesis of monensin A involves construction of a polyene polyketide backbone, subsequent epoxidation of the alkenes, and, lastly, formation of cyclic ethers via epoxide-opening cyclization. MonCI, a flavin-dependent monooxygenase, is thought to transform all three alkenes in the intermediate polyketide premonensin A into epoxides. Our crystallographic study has revealed that MonCI's exquisite stereocontrol is due to the preorganization of the active site residues which allows only one specific face of the alkene to approach the reactive C(4a)-hydroperoxyflavin moiety. Furthermore, MonCI has an unusually large substrate-binding cavity that can accommodate premonensin A in an extended or folded conformation which allows any of the three alkenes to be placed next to C(4a)-hydroperoxyflavin. MonCI, with its ability to perform multiple epoxidations on the same substrate in a stereospecific manner, demonstrates the extraordinary versatility of the flavin-dependent monooxygenase family of enzymes.

Structural and functional analyses of the echinomycin resistance conferring protein Ecm16 from Streptomyces lasalocidi.

Echinomycin is a natural product DNA bisintercalator antibiotic. The echinomycin biosynthetic gene cluster in Streptomyces lasalocidi includes a gene encoding the self-resistance protein Ecm16. Here, we present the 2.0 Å resolution crystal structure of Ecm16 bound to adenosine diphosphate. The structure of Ecm16 closely resembles that of UvrA, the DNA damage sensor component of the prokaryotic nucleotide excision repair system, but Ecm16 lacks the UvrB-binding domain and its associated zinc-binding module found in UvrA. Mutagenesis study revealed that the insertion domain of Ecm16 is required for DNA binding. Furthermore, the specific amino acid sequence of the insertion domain allows Ecm16 to distinguish echinomycin-bound DNA from normal DNA and link substrate binding to ATP hydrolysis activity. Expression of ecm16 in the heterologous host Brevibacillus choshinensis conferred resistance against echinomycin and other quinomycin antibiotics, including thiocoraline, quinaldopeptin, and sandramycin. Our study provides new insight into how the producers of DNA bisintercalator antibiotics fend off the toxic compounds that they produce.

The UvrA-like protein Ecm16 requires ATPase activity to render resistance against echinomycin.

Bacteria use various strategies to become antibiotic resistant. The molecular details of these strategies are not fully understood. We can increase our understanding by investigating the same strategies found in antibiotic-producing bacteria. In this work, we characterize the self-resistance protein Ecm16 encoded by echinomycin-producing bacteria. Ecm16 is a structural homolog of the nucleotide excision repair protein UvrA. Expression of ecm16 in the heterologous system Escherichia coli was sufficient to render resistance against echinomycin. Ecm16 binds DNA (double-stranded and single-stranded) using a nucleotide-independent binding mode. Ecm16's binding affinity for DNA increased by 1.7-fold when the DNA is intercalated with echinomycin. Ecm16 can render resistance against echinomycin toxicity independently of the nucleotide excision repair system. Similar to UvrA, Ecm16 has ATPase activity, and this activity is essential for Ecm16's ability to render echinomycin resistance. Notably, UvrA and Ecm16 were unable to complement each other's function. Together, our findings identify new mechanistic details of how a refurbished DNA repair protein Ecm16 can specifically render resistance to the DNA intercalator echinomycin.

Modular polyketide synthase contains two reaction chambers that operate asynchronously.

Type I modular polyketide synthases are homodimeric multidomain assembly line enzymes that synthesize a variety of polyketide natural products by performing polyketide chain extension and ß-keto group modification reactions. We determined the 2.4-angstrom-resolution x-ray crystal structure and the 3.1-angstrom-resolution cryo­electron microscopy structure of the Lsd14 polyketide synthase, stalled at the transacylation and condensation steps, respectively. These structures revealed how the constituent domains are positioned relative to each other, how they rearrange depending on the step in the reaction cycle, and the specific interactions formed between the domains. Like the evolutionarily related mammalian fatty acid synthase, Lsd14 contains two reaction chambers, but only one chamber in Lsd14 has the full complement of catalytic domains, indicating that only one chamber produces the polyketide product at any given time.

Two-Dimensional Design Strategy to Construct Smart Fluorescent Probes for the Precise Tracking of Senescence.

The tracking of cellular senescence usually depends on the detection of senescence-associated ß-galactosidase (SA-ß-gal). Previous probes for SA-ß-gal with this purpose only cover a single dimension: the accumulation of this enzyme in lysosomes. However, this is insufficient to determine the destiny of senescence because endogenous ß-gal enriched in lysosomes is not only related to senescence, but also to some other physiological processes. To address this issue, we introduce our fluorescent probes including a second dimension: lysosomal pH, since de-acidification is a unique feature of the lysosomes in senescent cells. With this novel design, our probes achieved excellent discrimination of SA-ß-gal from cancer-associated ß-gal, which enables them to track cellular senescence as well as tissue aging more precisely. Our crystal structures of a model enzyme E. coli ß-gal mutant (E537Q) complexed with each probe further revealed the structural basis for probe recognition.

First-generation species-selective chemical probes for fluorescence imaging of human senescence-associated ß-galactosidase.

Human senescence-associated ß-galactosidase (SA-ß-gal), the most widely used biomarker of aging, is a valuable tool for assessing the extent of cell 'healthy aging' and potentially predicting the health life span of an individual. Human SA-ß-gal is an endogenous lysosomal enzyme expressed from GLB1, the catalytic domain of which is very different from that of E. coli ß-gal, a bacterial enzyme encoded by lacZ. However, existing chemical probes for this marker still lack the ability to distinguish human SA-ß-gal from ß-gal of other species, such as bacterial ß-gal, which can yield false positive signals. Here, we show a molecular design strategy to construct fluorescent probes with the above ability with the aid of structure-based steric hindrance adjustment catering to different enzyme pockets. The resulting probes normally work as traditional SA-ß-gal probes, but they are unique in their powerful ability to distinguish human SA-ß-gal from E. coli ß-gal, thus achieving species-selective visualization of human SA-ß-gal for the first time. NIR-emitting fluorescent probe KSL11 as their representative further displays excellent species-selective recognition performance in biological systems, which has been herein verified by testing in senescent cells, in lacZ-transfected cells and in E. coli-ß-gal-contaminated tissue sections of mice. Because of our probes, it was also discovered that SA-ß-gal content in mice increased gradually with age and SA-ß-gal accumulated most in the kidneys among the main organs of naturally aging mice, suggesting that the kidneys are the organs with the most severe aging during natural aging.

De Novo Design and Implementation of a Tandem Acyl Carrier Protein Domain in a Type I Modular Polyketide Synthase.

During polyketide and fatty acid biosynthesis, the growing acyl chain is attached to the acyl carrier protein via a thioester linkage. The acyl carrier protein interacts with other enzymes that perform chain elongation and chain modification on the bound acyl chain. Most type I polyketide synthases and fatty acid synthases contain only one acyl carrier protein. However, polyunsaturated fatty acid synthases from deep-sea bacteria contain anywhere from two to nine acyl carrier proteins. Recent studies have shown that this tandem acyl carrier protein feature is responsible for the unusually high fatty acid production rate of deep-sea bacteria. To investigate if a similar strategy can be used to increase the production rate of type I polyketide synthases, a 3×ACP domain was rationally designed and genetically installed in module 6 of 6-deoxyerythronolide B synthase, which is a prototypical type I modular polyketide synthase that naturally harbors just one acyl carrier protein. This modification resulted in an ∼2.5-fold increase in the total amount of polyketide produced in vitro, demonstrating that installing a tandem acyl carrier domain in a type I polyketide synthase is an effective strategy for enhancing the rate of polyketide natural product biosynthesis.

Therapeutic applications of synthetic nucleic acid aptamers.

It is possible to generate oligonucleotide aptamers for a wide variety of target molecules using a process known as Systematic Evolution of Ligands by Exponential Enrichment. Researchers have successfully generated aptamers which recognize specific metal ions, small chemical compounds, peptides, proteins, saccharides, and even whole cells. Aptamers show much promise as future therapeutics and as drug targeting agents. A particularly active area of aptamer research in the past two years was development of aptamer based cancer therapeutics and development of aptamer based cancer drug delivery systems. Aptamers were also used to address inflammatory diseases, infectious diseases, cardiovascular diseases, and eye diseases.

Unraveling the structural basis for the unusually rich association of human leukocyte antigen DQ2.5 with class-II-associated invariant chain peptides.

Human leukocyte antigen (HLA)-DQ2.5 (DQA1*05/DQB1*02) is a class-II major histocompatibility complex protein associated with both type 1 diabetes and celiac disease. One unusual feature of DQ2.5 is its high class-II-associated invariant chain peptide (CLIP) content. Moreover, HLA-DQ2.5 preferentially binds the non-canonical CLIP2 over the canonical CLIP1. To better understand the structural basis of HLA-DQ2.5's unusual CLIP association characteristics, better insight into the HLA-DQ2.5·CLIP complex structures is required. To this end, we determined the X-ray crystal structure of the HLA-DQ2.5· CLIP1 and HLA-DQ2.5·CLIP2 complexes at 2.73 and 2.20 Å, respectively. We found that HLA-DQ2.5 has an unusually large P4 pocket and a positively charged peptide-binding groove that together promote preferential binding of CLIP2 over CLIP1. An α9-α22-α24-α31-ß86-ß90 hydrogen bond network located at the bottom of the peptide-binding groove, spanning from the P1 to P4 pockets, renders the residues in this region relatively immobile. This hydrogen bond network, along with a deletion mutation at α53, may lead to HLA-DM insensitivity in HLA-DQ2.5. A molecular dynamics simulation experiment reported here and recent biochemical studies by others support this hypothesis. The diminished HLA-DM sensitivity is the likely reason for the CLIP-rich phenotype of HLA-DQ2.5.

Epoxide hydrolase-lasalocid a structure provides mechanistic insight into polyether natural product biosynthesis.

Biosynthesis of some polyether natural products involves a kinetically disfavored epoxide-opening cyclic ether formation, a reaction termed anti-Baldwin cyclization. One such example is the biosynthesis of lasalocid A, an ionophore antibiotic polyether. During lasalocid A biosynthesis, an epoxide hydrolase, Lsd19, converts the bisepoxy polyketide intermediate into the tetrahydrofuranyl-tetrahydropyran product. We report the crystal structure of Lsd19 in complex with lasalocid A. The structure unambiguously shows that the C-terminal domain of Lsd19 catalyzes the intriguing anti-Baldwin cyclization. We propose a general mechanism for epoxide selection by ionophore polyether epoxide hydrolases.

Conversion of a disulfide bond into a thioacetal group during echinomycin biosynthesis.

Echinomycin is a nonribosomal depsipeptide natural product with a range of interesting bioactivities that make it an important target for drug discovery and development. It contains a thioacetal bridge, a unique chemical motif derived from the disulfide bond of its precursor antibiotic triostin A by the action of an S-adenosyl-L-methionine-dependent methyltransferase, Ecm18. The crystal structure of Ecm18 in complex with its reaction products S-adenosyl-L-homocysteine and echinomycin was determined at 1.50 Å resolution. Phasing was achieved using a new molecular replacement package called AMPLE, which automatically derives search models from structure predictions based on ab initio protein modelling. Structural analysis indicates that a combination of proximity effects, medium effects, and catalysis by strain drives the unique transformation of the disulfide bond into the thioacetal linkage.

Engineering the polyproline II propensity of a class II major histocompatibility complex ligand peptide.

Our immune system constantly samples peptides found inside the body as a means to detect foreign pathogens, infected cells, and tumorous cells. T cells, which carry out the critical task of distinguishing self from nonself peptides, can only survey peptides that are presented by the major histocompatibility complex protein. We investigated how the secondary structure of a peptide, namely, the polyproline II helix content, influences major histocompatibility complex binding. We synthesized 12 analogues of the wheat gluten derived α-I-gliadin peptide and tested their binding to the celiac disease associated HLA-DQ2 protein. Our analogue library represents a broad spectrum of polyproline II propensities, ranging from random coil structure to high polyproline II helix content. Overall, there was no noticeable correlation between the peptide polyproline II helix content and HLA-DQ2 binding. One analogue peptide, which has low polyproline II helix content, showed a 4.5-fold superior binding compared to native α-I-gliadin.

Enzymatic catalysis of anti-Baldwin ring closure in polyether biosynthesis.

Polycyclic polyether natural products have fascinated chemists and biologists alike owing to their useful biological activity, highly complex structure and intriguing biosynthetic mechanisms. Following the original proposal for the polyepoxide origin of lasalocid and isolasalocid and the experimental determination of the origins of the oxygen and carbon atoms of both lasalocid and monensin, a unified stereochemical model for the biosynthesis of polyether ionophore antibiotics was proposed. The model was based on a cascade of nucleophilic ring closures of postulated polyepoxide substrates generated by stereospecific oxidation of all-trans polyene polyketide intermediates. Shortly thereafter, a related model was proposed for the biogenesis of marine ladder toxins, involving a series of nominally disfavoured anti-Baldwin, endo-tet epoxide-ring-opening reactions. Recently, we identified Lsd19 from the Streptomyces lasaliensis gene cluster as the epoxide hydrolase responsible for the epoxide-opening cyclization of bisepoxyprelasalocid A to form lasalocid A. Here we report the X-ray crystal structure of Lsd19 in complex with its substrate and product analogue to provide the first atomic structure-to our knowledge-of a natural enzyme capable of catalysing the disfavoured epoxide-opening cyclic ether formation. On the basis of our structural and computational studies, we propose a general mechanism for the enzymatic catalysis of polyether natural product biosynthesis.

Structural and functional studies of trans-encoded HLA-DQ2.3 (DQA1*03:01/DQB1*02:01) protein molecule.

MHC class II molecules are composed of one α-chain and one ß-chain whose membrane distal interface forms the peptide binding groove. Most of the existing knowledge on MHC class II molecules comes from the cis-encoded variants where the α- and ß-chain are encoded on the same chromosome. However, trans-encoded class II MHC molecules, where the α- and ß-chain are encoded on opposite chromosomes, can also be expressed. We have studied the trans-encoded class II HLA molecule DQ2.3 (DQA1*03:01/DQB1*02:01) that has received particular attention as it may explain the increased risk of certain individuals to type 1 diabetes. We report the x-ray crystal structure of this HLA molecule complexed with a gluten epitope at 3.05 Å resolution. The gluten epitope, which is the only known HLA-DQ2.3-restricted epitope, is preferentially recognized in the context of the DQ2.3 molecule by T-cell clones of a DQ8/DQ2.5 heterozygous celiac disease patient. This preferential recognition can be explained by improved HLA binding as the epitope combines the peptide-binding motif of DQ2.5 (negative charge at P4) and DQ8 (negative charge at P1). The analysis of the structure of DQ2.3 together with all other available DQ crystal structures and sequences led us to categorize DQA1 and DQB1 genes into two groups where any α-chain and ß-chain belonging to the same group are expected to form a stable heterodimer.

T-cell response to gluten in patients with HLA-DQ2.2 reveals requirement of peptide-MHC stability in celiac disease.

BACKGROUND & AIMS: Celiac disease is a diet-induced, T cell-mediated enteropathy. The HLA variant DQ2.5 increases risk of the disease, and the homologous DQ2.2 confers a lower level of risk. As many as 5% of patients with celiac disease carry DQ2.2 without any other risk alleles. Epitopes commonly recognized by T cells of patients with HLA-DQ2.5 bind stably to DQ2.5 but unstably to DQ2.2. We investigated the response to gluten in patients with HLA-DQ2.2. METHODS: We generated intestinal T-cell lines and clones from 7 patients with HLA-DQ2.2 (but not DQ2.5) and characterized the responses of the cells to gluten. The epitope off-rate was evaluated by gel filtration and T cell-based assays. Peptide binding to DQ2.2 was studied with peptide substitutes and DQ2 mutants. RESULTS: Patients with DQ2.2 and no other risk alleles had gluten-reactive T cells that did not respond to the common DQ2.5-restricted T-cell epitopes. Instead, many of the T cells responded to a distinct epitope that was not recognized by those from patients with HLA-DQ2.5. This immunodominant epitope bound stably to DQ2.2. A serine residue at P3 was required for the stable binding. The effect of this residue related to a polymorphism at DQα22 that was previously shown to determine stable binding of peptides to DQ2.5. CONCLUSIONS: High levels of kinetic stability of peptide-major histocompatibility complexes are required to generate T-cell responses to gluten in celiac disease; the lower risk from DQ2.2 relates to constraints imposed on gluten peptides to stably bind this HLA molecule. These observations increase our understanding of the role of the major histocompatibility complex in determining T-cell responses in patients with celiac disease and are important for peptide-based vaccination strategies.

Siderophore-mediated iron acquisition in Bacillus anthracis and related strains.

Recent observations have shed light on some of the endogenous iron-acquisition mechanisms of members of the Bacillus cereus sensu lato group. In particular, pathogens in the B. cereus group use siderophores with both unique chemical structures and biological roles. This review will focus on recent discoveries in siderophore biosynthesis and biology in this group, which contains numerous human pathogens, most notably the causative agent of anthrax, Bacillus anthracis.

Differences in the risk of celiac disease associated with HLA-DQ2.5 or HLA-DQ2.2 are related to sustained gluten antigen presentation.

Celiac disease driven by an antigluten T cell response is strongly associated with the histocompatibility antigen HLA-DQ2.5 but is barely associated with HLA-DQ2.2. Yet these molecules have very similar peptide-binding motifs and both present gluten T cell epitopes. We found that DQ2.5(+) antigen-presenting cells (APCs) had greater stability of bound peptides and protracted gluten presentation relative to that of DQ2.2(+) cells. The improved ability of DQ2.5 to retain its peptide cargo can be ascribed to a polymorphism of DQalpha22 whereby DQ2.5 (tyrosine) can establish a hydrogen bond to the peptide main chain but DQ2.2 (phenylalanine) cannot. Our findings suggest that the kinetic stability of complexes of peptide and major histocompatibility complex (MHC) is of importance for the association of HLA with disease.

The missing link in petrobactin biosynthesis: asbF encodes a (-)-3-dehydroshikimate dehydratase.

The siderophore petrobactin harbors unique 3,4-dihydroxybenzoyl iron-liganding groups. These moieties are known to be synthesized from shikimate pathway precursors, but no reports of the biosynthetic enzymes responsible for this conversion have been published. The gene encoding AsbF from Bacillus thuringiensis 97-27 was overexpressed in an Escherichia coli host. AsbF rapidly and efficiently transforms (-)-3-dehydroshikimate (DHS) into 3,4-dihydroxybenzoate (k(cat)(DHS) = 217 +/- 10 min(-1); K(m)(DHS) = 125 +/- 14 microM) at 37 degrees C and has an absolute requirement for divalent metal. Finally, the pH versus k(cat)(DHS) profile revealed two ionizable groups (pK(a1) = 7.9 +/- 0.1, and pK(a2) = 9.3 +/- 0.1).