Structural Insight into the DNA Binding Function of Transcription Factor ERF.

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.

Discovery of a Cryptic Intermediate in Late Steps of Mithramycin Biosynthesis.

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 ß 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.

Structures of the Catalytic Domain of Bacterial Primase DnaG in Complexes with DNA Provide Insight into Key Priming Events.

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.

Structures of mithramycin analogues bound to DNA and implications for targeting transcription factor FLI1.

Transcription factors have been considered undruggable, but this paradigm has been recently challenged. DNA binding natural product mithramycin (MTM) is a potent antagonist of oncogenic transcription factor EWS-FLI1. Structural details of MTM recognition of DNA, including the FLI1 binding sequence GGA(A/T), are needed to understand how MTM interferes with EWS-FLI1. We report a crystal structure of an MTM analogue MTM SA-Trp bound to a DNA oligomer containing a site GGCC, and two structures of a novel analogue MTM SA-Phe in complex with DNA. MTM SA-Phe is bound to sites AGGG and GGGT on one DNA, and to AGGG and GGGA(T) (a FLI1 binding site) on the other, revealing how MTM recognizes different DNA sequences. Unexpectedly, at sub-micromolar concentrations MTMs stabilize FLI1-DNA complex on GGAA repeats, which are critical for the oncogenic function of EWS-FLI1. We also directly demonstrate by nuclear magnetic resonance formation of a ternary FLI1-DNA-MTM complex on a single GGAA FLI1/MTM binding site. These biochemical and structural data and a new FLI1-DNA structure suggest that MTM binds the minor groove and perturbs FLI1 bound nearby in the major groove. This ternary complex model may lead to development of novel MTM analogues that selectively target EWS-FLI1 or other oncogenic transcription factors, as anti-cancer therapeutics.

Mismatch repair genes Mlh1 and Mlh3 modify CAG instability in Huntington's disease mice: genome-wide and candidate approaches.

The Huntington's disease gene (HTT) CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington's disease Hdh(Q111) mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.Hdh(Q111) ) than on a 129 background (129.Hdh(Q111) ). Linkage mapping in (B6x129).Hdh(Q111) F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene Mlh1 as the most likely candidate modifier. Crossing B6.Hdh(Q111) mice onto an Mlh1 null background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. Hdh(Q111) somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1-MLH3) complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSß (MSH2-MSH3). The Mlh1 locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1 protein levels play an important role in driving of the efficiency of somatic expansions.

Structural Basis for Dimerization and DNA Binding of Transcription Factor FLI1.

FLI1 (Friend leukemia integration 1) is a metazoan transcription factor that is upregulated in a number of cancers. In addition, rearrangements of the fli1 gene cause sarcomas, leukemias, and lymphomas. These rearrangements encode oncogenic transcription factors, in which the DNA binding domain (DBD or ETS domain) of FLI1 on the C-terminal side is fused to a part of an another protein on the N-terminal side. Such abnormal cancer cell-specific fusions retain the DNA binding properties of FLI1 and acquire non-native protein-protein or protein-nucleic acid interactions of the substituted region. As a result, these fusions trigger oncogenic transcriptional reprogramming of the host cell. Interactions of FLI1 fusions with other proteins and with itself play a critical role in the oncogenic regulatory functions, and they are currently under intense scrutiny, mechanistically and as potential novel anticancer drug targets. We report elusive crystal structures of the FLI1 DBD, alone and in complex with cognate DNA containing a GGAA recognition sequence. Both structures reveal a previously unrecognized dimer of this domain, consistent with its dimerization in solution. The homodimerization interface is helix-swapped and dominated by hydrophobic interactions, including those between two interlocking Phe362 residues. A mutation of Phe362 to an alanine disrupted the propensity of this domain to dimerize without perturbing its structure or the DNA binding function, consistent with the structural observations. We propose that FLI1 DBD dimerization plays a role in transcriptional activation and repression by FLI1 and its fusions at promoters containing multiple FLI1 binding sites.

Structural insight into MtmC, a bifunctional ketoreductase-methyltransferase involved in the assembly of the mithramycin trisaccharide chain.

More and more post-PKS tailoring enzymes are recognized as being multifunctional and codependent on other tailoring enzymes. One of the recently discovered intriguing examples is MtmC, a bifunctional TDP-4-keto-d-olivose ketoreductase-methyltransferase, which-in codependence with glycosyltransferase MtmGIV-is a key contributor to the biosynthesis of the critical trisaccharide chain of the antitumor antibiotic mithramycin (MTM), produced by Streptomyces argillaceus. We report crystal structures of three binary complexes of MtmC with its methylation cosubstrate SAM, its coproduct SAH, and a nucleotide TDP as well as crystal structures of two ternary complexes, MtmC-SAH-TDP-4-keto-d-olivose and MtmC-SAM-TDP, in the range of 2.2-2.7 Å resolution. The structures reveal general and sugar-specific recognition and catalytic structural features of MtmC. Depending on the catalytic function that is conducted by MtmC, it must bind either NADPH or SAM in the same cofactor binding pocket. A tyrosine residue (Tyr79) appears as a lid covering the sugar moiety of the substrate during the methyl transfer reaction. This residue swings out of the active site by ~180° in the absence of the substrate. This unique conformational change likely serves to release the methylated product and, possibly, to open the active site for binding the bulkier cosubstrate NADPH prior to the reduction reaction.

Crystal structure of O-methyltransferase CalO6 from the calicheamicin biosynthetic pathway: a case of challenging structure determination at low resolution.

BACKGROUND: Calicheamicins (CAL) are enedyine natural products with potent antibiotic and cytotoxic activity, used in anticancer therapy. The O-methyltransferase CalO6 is proposed to catalyze methylation of the hydroxyl moiety at the C2 position of the orsellinic acid group of CAL. RESULTS: Crystals of CalO6 diffracted non-isotropically, with the usable data extending to 3.4 Å. While no single method of crystal structure determination yielded a structure of CalO6, we were able to determine its structure by using molecular replacement-guided single wavelength anomalous dispersion by using diffraction data from native crystals of CalO6 and a highly non-isomorphous mercury derivative. The structure of CalO6 reveals the methyltransferase fold and dimeric organization characteristic of small molecule O-methyltransferases involved in secondary metabolism in bacteria and plants. Uncommonly, CalO6 was crystallized in the absence of S-adenosylmethionine (SAM; the methyl donor) or S-adenosylhomocysteine (SAH; its product). CONCLUSIONS: Likely as a consequence of the dynamic nature of CalO6 in the absence of its cofactor, the central region of CalO6, which forms a helical lid-like structure near the active site in CalO6 and similar enzymes, is not observed in the electron density. We propose that this region controls the entry of SAM into and the exit of SAH from the active site of CalO6 and shapes the active site for substrate binding and catalysis.

Coordinated processing of 3' slipped (CAG)n/(CTG)n hairpins by DNA polymerases ß and δ preferentially induces repeat expansions.

Expansion of CAG/CTG trinucleotide repeats causes certain familial neurological disorders. Hairpin formation in the nascent strand during DNA synthesis is considered a major path for CAG/CTG repeat expansion. However, the underlying mechanism is unclear. We show here that removal or retention of a nascent strand hairpin during DNA synthesis depends on hairpin structures and types of DNA polymerases. Polymerase (pol) δ alone removes the 3'-slipped hairpin using its 3'-5' proofreading activity when the hairpin contains no immediate 3' complementary sequences. However, in the presence of pol ß, pol δ preferentially facilitates hairpin retention regardless of hairpin structures. In this reaction, pol ß incorporates several nucleotides to the hairpin 3'-end, which serves as an effective primer for the continuous DNA synthesis by pol δ, thereby leading to hairpin retention and repeat expansion. These findings strongly suggest that coordinated processing of 3'-slipped (CAG)n/(CTG)n hairpins by polymerases δ and ß on during DNA synthesis induces CAG/CTG repeat expansions.

The Werner syndrome protein promotes CAG/CTG repeat stability by resolving large (CAG)(n)/(CTG)(n) hairpins.

Expansion of CAG/CTG repeats causes certain neurological and neurodegenerative disorders, and the formation and subsequent persistence of stable DNA hairpins within these repeats are believed to contribute to CAG/CTG repeat instability. Human cells possess a DNA hairpin repair (HPR) pathway, which removes various (CAG)(n) and (CTG)(n) hairpins in a nick-directed and strand-specific manner. Interestingly, this HPR system processes a (CTG)(n) hairpin on the template DNA strand much less efficiently than a (CAG)(n) hairpin on the same strand (Hou, C., Chan, N. L., Gu, L., and Li, G. M. (2009) Incision-dependent and error-free repair of (CAG)(n)/(CTG)(n) hairpins in human cell extracts. Nat. Struct. Mol. Biol. 16, 869-875), suggesting the involvement of an additional component for (CTG)(n) HPR. To identify this activity, a functional in vitro HPR assay was used to screen partially purified HeLa nuclear fractions for their ability to stimulate (CTG)(n) HPR. We demonstrate here that the stimulating activity is the Werner syndrome protein (WRN). Although WRN contains both a 3'→5' helicase activity and a 3'→5' exonuclease activity, the stimulating activity was found to be the helicase activity, as a WRN helicase mutant failed to enhance (CTG)(n) HPR. Consistently, WRN efficiently unwound large (CTG)(n) hairpins and promoted DNA polymerase δ-catalyzed DNA synthesis using a (CTG)(n) hairpin as a template. We, therefore, conclude that WRN stimulates (CTG)(n) HPR on the template DNA strand by resolving the hairpin so that it can be efficiently used as a template for repair or replicative synthesis.

Mithplatins: Mithramycin SA-Pt(II) Complex Conjugates for the Treatment of Platinum-Resistant Ovarian Cancers.

DNA coordinating platinum (Pt) containing compounds cisplatin and carboplatin have been used for the treatment of ovarian cancer therapy for four decades. However, recurrent Pt-resistant cancers are a major cause of mortality. To combat Pt-resistant ovarian cancers, we designed and synthesized a conjugate of an anticancer drug mithramycin with a reactive Pt(II) bearing moiety, which we termed mithplatin. The conjugates displayed both the Mg2+ -dependent noncovalent DNA binding characteristic of mithramycin and the covalent crosslinking to DNA of the Pt. The conjugate was three times as potent as cisplatin against ovarian cancer cells. The DNA lesions caused by the conjugate led to the generation of DNA double-strand breaks, as also observed with cisplatin. Nevertheless, the conjugate was highly active against both Pt-sensitive and Pt-resistant ovarian cancer cells. This study paves the way to developing mithplatins to combat Pt-resistant ovarian cancers.

Leveraging User Comments for the Construction of Recycled Water Infrastructure-Evidence from an Eye-Tracking Experiment.

Building sufficient recycled water infrastructure is an effective way to solve problems related to water shortages and environmental degradation, and is of great strategic significance for saving resources, protecting the ecological environment, and promoting sustainable social and economic development. Although recycled water is environmentally friendly, the public is still skeptical about its use, which has led to the failure of a large number of recycled water infrastructure investments; therefore, increasing the public's willingness to re-use is critical for the construction of recycled water infrastructure. To identify the influence mechanism of user comments on public re-use behaviors, we conducted an eye-tracking experiment in China. The results demonstrated that (1) perceived usefulness, perceived quality, and perceived risk have significant impacts on the public's willingness to buy; (2) user reviews can enhance the public's perceived usefulness of recycled products and increase their willingness to buy; and (3) in the process of consumption, the public tends to pay attention to negative reviews, where user reviews alter the perceived risks and perceived prices of recycled products, thereby affecting the willingness to buy of consumers. This study provides a scientific reference for the construction of recycled water infrastructure and the further promotion of recycled water.

Allosteric interference in oncogenic FLI1 and ERG transactions by mithramycins.

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 (Cbfß), 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.

Development of Single-Stranded DNA Bisintercalating Inhibitors of Primase DnaG as Antibiotics.

Many essential enzymes in bacteria remain promising potential targets of antibacterial agents. In this study, we discovered that dequalinium, a topical antibacterial agent, is an inhibitor of Staphylococcus aureus primase DnaG (SaDnaG) with low-micromolar minimum inhibitory concentrations against several S. aureus strains, including methicillin-resistant bacteria. Mechanistic studies of dequalinium and a series of nine of its synthesized analogues revealed that these compounds are single-stranded DNA bisintercalators that penetrate a bacterium by compromising its membrane. The best compound of this series likely interacts with DnaG directly, inhibits both staphylococcal cell growth and biofilm formation, and displays no significant hemolytic activity or toxicity to mammalian cells. This compound is an excellent lead for further development of a novel anti-staphylococcal therapeutic.

Alteration of MDM2 by the Small Molecule YF438 Exerts Antitumor Effects in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) exhibits a high mortality rate and is the most aggressive subtype of breast cancer. As previous studies have shown that histone deacetylases (HDAC) may represent molecular targets for TNBC treatment, we screened a small library of synthetic molecules and identified a potent HDAC inhibitor (HDACi), YF438, which exerts effective anti-TNBC activity both in vitro and in vivo. Proteomic and biochemical studies revealed that YF438 significantly downregulated mouse double minute 2 homolog (MDM2) expression. In parallel, loss of MDM2 expression or blocking MDM2 E3 ligase activity rendered TNBC cells less sensitive to YF438 treatment, revealing an essential role of MDM2 E3 ligase activity in YF438-induced inhibition of TNBC. Mechanistically, YF438 disturbed the interaction between HDAC1 and MDM2, induced the dissociation of MDM2-MDMX, and subsequently increased MDM2 self-ubiquitination to accelerate its degradation, which ultimately inhibited growth and metastasis of TNBC cells. In addition, analysis of clinical tissue samples demonstrated high expression levels of MDM2 in TNBC, and MDM2 protein levels closely correlated with TNBC progression and metastasis. Collectively, these findings show that MDM2 plays an essential role in TNBC progression and targeting the HDAC1-MDM2-MDMX signaling axis with YF438 may provide a promising therapeutic option for TNBC. Furthermore, this novel underlying mechanism of a hydroxamate-based HDACi in altering MDM2 highlights the need for further development of HDACi for TNBC treatment. SIGNIFICANCE: This study uncovers the essential role of MDM2 in TNBC progression and suggests that targeting the HDAC1-MDM2-MDMX axis with a hydroxamate-based HDACi could be a promising therapeutic strategy for TNBC.

Structure-based design of haloperidol analogues as inhibitors of acetyltransferase Eis from Mycobacterium tuberculosis to overcome kanamycin resistance.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a deadly bacterial disease. Drug-resistant strains of Mtb make eradication of TB a daunting task. Overexpression of the enhanced intracellular survival (Eis) protein by Mtb confers resistance to the second-line antibiotic kanamycin (KAN). Eis is an acetyltransferase that acetylates KAN, inactivating its antimicrobial function. Development of Eis inhibitors as KAN adjuvant therapeutics is an attractive path to forestall and overcome KAN resistance. We discovered that an antipsychotic drug, haloperidol (HPD, 1), was a potent Eis inhibitor with IC50 = 0.39 ± 0.08 µM. We determined the crystal structure of the Eis-haloperidol (1) complex, which guided synthesis of 34 analogues. The structure-activity relationship study showed that in addition to haloperidol (1), eight analogues, some of which were smaller than 1, potently inhibited Eis (IC50 ≤ 1 µM). Crystal structures of Eis in complexes with three potent analogues and droperidol (DPD), an antiemetic and antipsychotic, were determined. Three compounds partially restored KAN sensitivity of a KAN-resistant Mtb strain K204 overexpressing Eis. The Eis inhibitors generally did not exhibit cytotoxicity against mammalian cells. All tested compounds were modestly metabolically stable in human liver microsomes, exhibiting 30-60% metabolism over the course of the assay. While direct repurposing of haloperidol as an anti-TB agent is unlikely due to its neurotoxicity, this study reveals potential approaches to modifying this chemical scaffold to minimize toxicity and improve metabolic stability, while preserving potent Eis inhibition.

Mismatch recognition protein MutSbeta does not hijack (CAG)n hairpin repair in vitro.

CAG repeats form stable hairpin structures, which are believed to be responsible for CAG repeat expansions associated with certain human neurological diseases. Human cells possess an accurate DNA hairpin repair system that prevents expansion of disease-associated CAG repeats. Based on transgenic animal studies, it is suggested that (CAG)(n) expansion is caused by abnormal binding of the MutSbeta mismatch recognition protein to (CAG)(n) hairpins, leading to hijacking mismatch repair function during (CAG)(n) hairpin repair. We demonstrate here that MutSbeta displays identical biochemical and biophysical activities (including ATP-provoked conformational change, ATPase, ATP binding, and ADP binding) when interacting with a (CAG)(n) hairpin and a mismatch. More importantly, our in vitro functional hairpin repair assays reveal that excess MutSbeta does not inhibit (CAG)(n) hairpin repair in HeLa nuclear extracts. Evidence presented here provides a novel view as to whether or not MutSbeta is involved in CAG repeat instability in humans.

The effect of recycled water information disclosure on public acceptance of recycled water-Evidence from residents of Xi'an, China.

Recycled water, the secondary water source of a city, is vital to alleviate regional water resource shortage and promote environmental conservation. The attitude and acceptance toward recycled water of the public, its direct user, hold the key to the implementation of a recycled water project. Currently, the public's low intention of using recycled water constitutes the biggest obstacle to its popularization. To identify the factors of public acceptance of recycled water and their influence path, this study analyzes the effect mechanism of information disclosure of recycled water on the public's acceptance of it based on the consciousness-context-behavior theory and by adopting a structural equation model. The results are as follows: (1) The public's consciousness of water saving, risk perception of recycled water, and consciousness of environmental responsibility can effectively promote public acceptance of recycled water; (2) The consciousness of water saving and that of environmental responsibility have a significant effect on public acceptance of recycled water, and so do the consciousness of water saving and the risk perception of recycled water; and (3) Recycled water information disclosure has the most significant regulatory effect on consciousness and public acceptance.

Utilizing guanine-coordinated Zn2+ ions to determine DNA crystal structures by single-wavelength anomalous diffraction.

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.

How mithramycin stereochemistry dictates its structure and DNA binding function.

An aureolic acid natural product mithramycin (MTM) has been known for its potent antineoplastic properties. MTM inhibits cell growth by binding in the minor groove of double-stranded DNA as a dimer, in which the two molecules of MTM are coordinated to each other through a divalent metal ion. A crystal structure of an MTM analogue, MTM SA-Phe, in the active metal ion-coordinated dimeric form demonstrates how the stereochemical features of MTM define the helicity of the dimeric scaffold for its binding to a right-handed DNA double helix. We also show crystallographically and biochemically that MTM, but not MTM SA-Phe, can be inactivated by boric acid through formation of a large macrocyclic species, in which two molecules of MTM are crosslinked to each other through 3-side chain-boron-sugar intermolecular bonds. We discuss these structural and biochemical properties in the context of MTM biosynthesis and the design of MTM analogues as anticancer therapeutics.