Covering: 2009 up to August 2023
Prenyltransferases (PTs) are involved in the primary and the secondary metabolism of plants, bacteria, and fungi, and they are key enzymes in the biosynthesis of many ...clinically relevant natural products (NPs). The continued biochemical and structural characterization of the soluble dimethylallyl tryptophan synthase (DMATS) PTs over the past two decades have revealed the significant promise that these enzymes hold as biocatalysts for the chemoenzymatic synthesis of novel drug leads. This is a comprehensive review of DMATSs describing the structure-function relationships that have shaped the mechanistic underpinnings of these enzymes, as well as the application of this knowledge to the engineering of DMATSs. We summarize the key findings and lessons learned from these studies over the past 14 years (2009-2023). In addition, we identify current gaps in our understanding of these fascinating enzymes.
This review unpacks the accumulated knowledge of the structural bases of the unique properties and capabilities of DMATS-type prenyltransferases (PTs) that make them promising biocatalysts.
Fluoroquinolone antibiotics are among the most potent second-line drugs used for treatment of multidrug-resistant tuberculosis (MDR TB), and resistance to this class of antibiotics is one criterion ...for defining extensively drug resistant tuberculosis (XDR TB). Fluoroquinolone resistance in Mycobacterium tuberculosis has been associated with modification of the quinolone resistance determining region (QRDR) of gyrA. Recent studies suggest that amino acid substitutions in gyrB may also play a crucial role in resistance, but functional genetic studies of these mutations in M. tuberculosis are lacking. In this study, we examined twenty six mutations in gyrase genes gyrA (seven) and gyrB (nineteen) to determine the clinical relevance and role of these mutations in fluoroquinolone resistance. Transductants or clinical isolates harboring T80A, T80A+A90G, A90G, G247S and A384V gyrA mutations were susceptible to all fluoroquinolones tested. The A74S mutation conferred low-level resistance to moxifloxacin but susceptibility to ciprofloxacin, levofloxacin and ofloxacin, and the A74S+D94G double mutation conferred cross resistance to all the fluoroquinolones tested. Functional genetic analysis and structural modeling of gyrB suggest that M330I, V340L, R485C, D500A, D533A, A543T, A543V and T546M mutations are not sufficient to confer resistance as determined by agar proportion. Only three mutations, N538D, E540V and R485C+T539N, conferred resistance to all four fluoroquinolones in at least one genetic background. The D500H and D500N mutations conferred resistance only to levofloxacin and ofloxacin while N538K and E540D consistently conferred resistance to moxifloxacin only. Transductants and clinical isolates harboring T539N, T539P or N538T+T546M mutations exhibited low-level resistance to moxifloxacin only but not consistently. These findings indicate that certain mutations in gyrB confer fluoroquinolone resistance, but the level and pattern of resistance varies among the different mutations. The results from this study provide support for the inclusion of the QRDR of gyrB in molecular assays used to detect fluoroquinolone resistance in M. tuberculosis.
ETS family transcription factors of ERG and FLI1 play a key role in oncogenesis of prostate cancer and Ewing sarcoma by binding regulatory DNA sites and interfering with function of other factors. ...Mithramycin (MTM) is an anti-cancer, DNA binding natural product that functions as a potent antagonist of ERG and FLI1 by an unknown mechanism. We present a series of crystal structures of the DNA binding domain (DBD) of ERG/FLI1 culminating in a structure of a high-order complex of the ERG/FLI1 DBD, transcription factor Runx2, core-binding factor beta (Cbfβ), and MTM on a DNA enhancer site, along with supporting DNA binding studies using MTM and its analogues. Taken together, these data provide insight into allosteric mechanisms underlying ERG and FLI1 transactions and their disruption by MTM analogues.
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•DNA binding data demonstrate how FLI1 and Cbfβ affect DNA affinity of Runx2•Structures of FLI1-DNA and FLI1-Runx2-DNA show that the Runx2-FLI1 interactions are allosteric•Mithramycin analogues have perturbing effects on the FLI1/ERG-Runx2-Cbfβ-DNA complex•The structure of MTM-ERG-Runx2-Cbfβ-DNA shows how MTM analogues interact with Runx2
Oncogenic protein fusions with FLI1 and ERG cause Ewing sarcoma and metastatic prostate cancer. In this study, Hou et al. elucidate how these proteins form oncogenic complexes on DNA and how analogues of the natural product mithramycin interfere with these complexes.
Interrupted adenylation domains are enigmatic fusions, in which one enzyme is inserted into another to form a highly unusual bifunctional enzyme. We present the first crystal structure of an ...interrupted adenylation domain that reveals a unique embedded methyltransferase. The structure and functional data provide insight into how these enzymes N-methylate amino acid precursors en route to nonribosomal peptides.
Nonribosomal peptides (NRPs) are natural products that are biosynthesized by large multi-enzyme assembly lines called nonribosomal peptide synthetases (NRPSs). We have previously discovered that ...backbone or side chain methylation of NRP residues is carried out by an interrupted adenylation (A) domain that contains an internal methyltransferase (M) domain, while maintaining a monolithic AMA fold of the bifunctional enzyme. A key question that has remained unanswered is at which step of the assembly line mechanism the methylation by these embedded M domains takes place. Does the M domain methylate an amino acid residue tethered to a thiolation (T) domain on same NRPS module (in cis), or does it methylate this residue on a nascent peptide tethered to a T domain on another module (in trans)? In this study, we investigated the kinetics of methylation by wild-type AMAT tridomains from two NRPSs involved in biosynthesis of anticancer depsipeptides thiocoraline and echinomycin, and by mutants of these domains, for which methylation can occur only in trans. The analysis of the methylation kinetics unequivocally demonstrated that the wild-type AMATs methylate overwhelmingly in cis, strongly suggesting that this is also the case in the context of the entire NRPS assembly line process. The mechanistic insight gained in this study will facilitate rational genetic engineering of NRPS to generate unnaturally methylated NRPs.
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•Methylation mechanism in nonribosomal peptide synthetases remains to be determined.•Kinetic studies unequivocally established methylation occurs within one module.•This mechanistic logic will aid in engineering unnaturally methylated peptides.
ETS family transcription factors control development of different cell types in humans, whereas deregulation of these proteins leads to severe developmental syndromes and cancers. One of a few ...members of the ETS family that are known to act solely as repressors, ERF, is required for normal osteogenesis and hematopoiesis. Another important function of ERF is acting as a tumor suppressor by antagonizing oncogenic fusions involving other ETS family factors. The structure of ERF and the DNA binding properties specific to this protein have not been elucidated. In this study, we determined two crystal structures of the complexes of the DNA binding domain of ERF with DNA. In one, ERF is in a distinct dimeric form, with Cys72 in a reduced state. In the other, two dimers of ERF are assembled into a tetramer that is additionally locked by two Cys72–Cys72 disulfide bonds across the dimers. In the tetramer, the ERF molecules are bound to a pseudocontinuous DNA on the same DNA face at two GGAA binding sites on opposite strands. Sedimentation velocity analysis showed that this tetrameric assembly forms on continuous DNA containing such tandem sites spaced by 7 bp. Our bioinformatic analysis of three previously reported sets of ERF binding loci across entire genomes showed that these loci were enriched in such 7 bp spaced tandem sites. Taken together, these results strongly suggest that the observed tetrameric assembly is a functional state of ERF in the human cell.
Bacterial primase DnaG is an essential nucleic acid polymerase that generates primers for replication of chromosomal DNA. The mechanism of DnaG remains unclear due to the paucity of structural ...information on DnaG in complexes with other replisome components. Here we report the first crystal structures of noncovalent DnaG–DNA complexes, obtained with the RNA polymerase domain of Mycobacterium tuberculosis DnaG and various DNA ligands. One structure, obtained with ds DNA, reveals interactions with DnaG as it slides on ds DNA and suggests how DnaG binds template for primer synthesis. In another structure, DNA in the active site of DnaG mimics the primer, providing insight into mechanisms for the nucleotide transfer and DNA translocation. In conjunction with the recent cryo-EM structure of the bacteriophage T7 replisome, this study yields a model for primer elongation and hand-off to DNA polymerase.
MtmOIV and MtmW catalyze the final two reactions in the mithramycin (MTM) biosynthetic pathway, the Baeyer–Villiger opening of the fourth ring of premithramycin B (PMB), creating the C3 pentyl side ...chain, strictly followed by reduction of the distal keto group on the new side chain. Unexpectedly this results in a C2 stereoisomer of mithramycin, iso‐mithramycin (iso‐MTM). Iso‐MTM undergoes a non‐enzymatic isomerization to MTM catalyzed by Mg2+ ions. Crystal structures of MtmW and its complexes with co‐substrate NADPH and PEG, suggest a catalytic mechanism of MtmW. The structures also show that a tetrameric assembly of this enzyme strikingly resembles the ring‐shaped β subunit of a vertebrate ion channel. We show that MtmW and MtmOIV form a complex in the presence of PMB and NADPH, presumably to hand over the unstable MtmOIV product to MtmW, yielding iso‐MTM, as a potential self‐resistance mechanism against MTM toxicity.
MtmOIV and MtmW catalyze the final two reactions in the mithramycin biosynthetic pathway, the Baeyer–Villiger opening of the fourth ring of premithramycin B, creating the C3 pentyl side chain, followed by reduction of the distal keto group on the new side chain. Unexpectedly this results in a C2 stereoisomer of mithramycin, iso‐mithramycin (iso‐MTM).
The experimental phase determination of crystal structures of nucleic acids and nucleic acid–ligand complexes would benefit from a facile method. Even for double‐stranded DNA, software‐generated ...models are generally insufficiently accurate to serve as molecular replacement search models, necessitating experimental phasing. Here, it is demonstrated that Zn2+ ions coordinated to the N7 atom of guanine bases generate sufficient anomalous signal for single‐wavelength anomalous diffraction (SAD) phasing of DNA crystal structures. Using zinc SAD, three crystal structures of double‐stranded DNA oligomers, 5′‐AGGGATCCCT‐3′, 5′‐GGGATCCC‐3′ and 5′‐GAGGCCTC‐3′, were determined. By determining the crystal structure of one of these oligomers, GAGGCCTC, in the presence of Mg2+ instead of Zn2+, it was demonstrated that Zn2+ is not structurally perturbing. These structures allowed the analysis of structural changes in the DNA on the binding of analogues of the natural product mithramycin to two of these oligomers, AGGGATCCCT and GAGGCCTC. Zinc SAD may become a routine approach for determining the crystal structures of nucleic acids and their complexes with small molecules.
Guanine N7–Zn2+ coordination served as the basis for the determination of three DNA crystal structures using zinc single‐wavelength anomalous diffraction. This method is likely to be broadly applicable to the experimental determination of crystal structures of nucleic acids.
Over one and a half million people die of tuberculosis (TB) each year. Multidrug-resistant TB infections are especially dangerous, and new drugs are needed to combat them. The high cost and ...complexity of drug development make repositioning of drugs that are already in clinical use for other indications a potentially time- and money-saving avenue. In this study, we identified among existing drugs five compounds: azelastine, venlafaxine, chloroquine, mefloquine, and proguanil as inhibitors of acetyltransferase Eis from Mycobacterium tuberculosis, a causative agent of TB. Eis upregulation is a cause of clinically relevant resistance of TB to kanamycin, which is inactivated by Eis-catalyzed acetylation. Crystal structures of these drugs as well as chlorhexidine in complexes with Eis showed that these inhibitors were bound in the aminoglycoside binding cavity, consistent with their established modes of inhibition with respect to kanamycin. Among three additionally synthesized compounds, a proguanil analogue, designed based on the crystal structure of the Eis–proguanil complex, was 3-fold more potent than proguanil. The crystal structures of these compounds in complexes with Eis explained their inhibitory potencies. These initial efforts in rational drug repositioning can serve as a starting point in further development of Eis inhibitors.
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