Martin Tanner


Relevant Degree Programs


Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - Nov 2019)
Design, synthesis and studies of guanidinium-based inhibitors for isoprenoid biosynthetic pathway enzymes (2019)

In this thesis an inhibition strategy was developed to target enzymes that utilize allylic diphosphates. Positively-charged inhibitors that mimic the transition states/intermediates formed with these enzymes were synthesized. In chapter two, inhibitor 2 containing a guanidinium moiety appended to a phosphonylphosphinate was designed to mimic the transition state for the dissociation of dimethylallyl diphosphate into an allylic carbocation-pyrophosphate ion-pair. To test for the effectiveness of incorporating a guanidinium functionality into inhibitors of human farnesyl diphosphate synthase, inhibitors 3 and 4 were also prepared. Inhibitor 3 has a positive charge localized onto one atom, and inhibitor 4 is isosteric to inhibitor 2, but lacks positive charge. The inhibitors displayed IC50 values that were significantly higher than the substrate Km value, indicating that the positive charge did not result in tight binding to the enzyme. We decided to apply our inhibition strategy on other allylic diphosphate utilizing-enzymes. In chapter three, inhibitors bearing a guanidinium/amidinium moiety appended to a phosphonylphosphinate and flanked by a hydrocarbon tail (inhibitors 26 and 36) were synthesized. A neutral inhibitor 34 was also prepared as control. These inhibitors were tested against human squalene synthase (HSQS) and bacterial dehydrosqualene synthase (DSQS) from Staphylococcus aureus. It was anticipated that the lipid chain might increase the enzyme’s affinity towards the inhibitors. The positively-charged inhibitors acted as competitive inhibitors (low micromolar KI values) against DSQS. Similar trends were observed for the first half reaction of HSQS. Surprisingly, the neutral inhibitor was the most potent for both enzymes. These results indicated that the active site of both enzymes does not directly stabilize the allylic carbocation. We reasoned that these positively-charged inhibitors might be effective with enzymes that generate carbocation intermediates that are not stabilized by resonance or ion-pair interactions. Such a strategy was applied in chapter four, where inhibitors 2 – 4 were tested against isopentenyl diphosphate isomerase (IDI), and acted as competitive inhibitors of IDI. Notably, inhibitor 2 bound 400 times more tightly than its neutral isostere, inhibitor 4. We reasoned that inhibitor 2 allows for proper positioning of the positive charge in the active site, leading to favorable electrostatic interactions.

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Prenyltransferases: Mechanistic Studies and Inhibitor Design (2016)

The cyclic dipeptide N-prenyltransferase (CdpNPT) catalyzes the reverse C-3 prenylation of a variety of cyclic dipeptides and benzodiazepinediones. A previous study misassigned the structure of the product of this reaction. In this work, the true product of the CdpNPT-catalyzed reaction between cyclo-L-Trp-L-Trp and dimethylallyl diphosphate (DMAPP) is identified as a C-3 reverse prenylated species. Furthermore, the non-enzymatic Cope/aza-Cope rearrangement of the CdpNPT product was examined under acidic conditions. Our results indicated that only the aza-Cope rearrangement onto the N-1 position of the indole ring can occur and no Cope rearrangement onto the C-4 position was observed. These results suggest that in the absence of an enzyme active site, the aza-Cope rearrangement is preferred over the Cope rearrangement. Brevianamide F prenyltransferase (FtmPT1) catalyzes the C-2 normal prenylation of brevianamide F (cyclo-L-Trp-L-Pro). A mechanism involving a direct C-2 attack was proposed for this reaction. However, the structural analysis of FtmPT1, as well as studies of alternate substrates and mutant enzymes suggested that a different mechanism involving an initial C-3 reverse prenylation followed by a rearrangement may be operative. In this work, we investigated the reactivity of FtmPT1 with tryptophan and cyclo-L-Trp-L-Trp, as well as two alternate substrates: 5-hydroxybrevianamide F and 2-methylbrevianamide F. The isolated products were reverse prenylated at C-3 and normal prenylated at N-1, C-2, C-3, or C-4. The formation of these products can be rationalized through mechanisms involving either an initial C-3 normal or C-3 reverse prenylation as the first step of catalysis. 4-Dimethylallyltryptophan synthase is an aromatic prenyltransferase that catalyzes an electrophilic aromatic substitution reaction between DMAPP and L-tryptophan. The reaction is believed to proceed via the dissociation of DMAPP to form a dimethylallyl cation/phosphate ion pair. An inhibitor containing a guanidinium moiety appended to a phosphorylated phosphonate was designed in order to mimic the transition state for the dissociation of DMAPP into an allylic carbocation and pyrophosphate. This compound was found to serve as a potent competitive inhibitor (submicromolar Ki value) of the enzyme 4-DMATS.

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Inhibition Studies of Glu Ligase TTLL7 and Peptidoglycan Peptidase Csd4 (2015)

Microtubules are one of the major components of the cytoskeleton, and are comprised of two kinds of tubulin proteins. The presence of diverse post-translational modifications provides microtubules with multiple biological functions, including both structural and physiological roles. Polyglutamylation is one of the widespread post-translational modifications and is catalyzed by the tubulin tyrosine ligase-like (TTLL) enzymes. The reaction adds multiple glutamates to the glutamate side chain near the tubulin C-terminus to form a polyglutamate chain of various lengths. Such a side chain can be recognized by microtubule-associated proteins (MAPs) as a regulation signal. Our work focuses on the inhibition studies of the enzyme TTLL7. Three inhibitors (Inhibitor 1 – 3) targeting different stages of polyglutamylation were designed and synthesized. The inhibitors were then tested in our collaborator Dr. Roll-Mecak’s Lab, and we found Inhibitor 2, that targets an elongation process, had the highest potency with an IC₅₀ of approximately 150 μM.Peptidoglycan is a key component of the bacterial cell wall and plays an essential role in determining bacterial cell morphology. The cell shape determining genes were recently discovered in Helicobacter pylori, which encode different DD-, DL- or endo-, carboxypeptidases. Theses peptidases hydrolyze the peptide bond in either crosslinked or uncrosslinked muropeptides in order to alter bacterial cell shape. Our study focuses on the enzyme Csd4, a carboxypeptidase that cleaves the PG tripeptides PG-L-Ala-iso-D-Glu-meso-Dap to the dipeptide PG-L-Ala-iso-D-Glu. Its homolog Pgp1 in Campylobacter jejuni was also identified recently. We synthesized a simple tripeptide Ac-L-Ala- iso-D-Glu-meso-Dap as a substrate for Csd4, which was successfully co-crystallized with the enzyme. We also measured the activity of Csd4 with this substrate. The Michaelis constant (KM) and catalytic rate constant (kcat) are 112 μM and 0.044 s-¹, respectively. Based on a mechanistic analysis, we designed and synthesized a pseudodipeptidyl phosphinate as a Csd4 inhibitor. The inhibitor was tested and gave a KI of 3.3 μM. The crystal structure of Csd4-inhibitor proved the supported mechanism involving ligation of the oxyanion tetrahedral intermediate by the zinc ion. In vivo studies with the inhibitor showed it induced significant cell straightening in H. pylori and acapsular C. jejuni at millimolar concentration.

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Mechanistic Studies on 4-Dimethylallyltryptophan Synthase and the N-Prenyltransferase Cymd (2015)

Prenylated Indole alkaloids comprise a large group of biologically active molecules that include the ergot alkaloids. Prenylation is often important for the activity of these compounds and is catalyzed by an emerging new class of enzyme, the indole prenyltransferases. These enzymes are metal independent and share a unique αββα fold. 4-Dimethylallyltryptophan synthase (DMATS) is an indole prenyltransferase that transfers the dimethylallyl group onto the C-4 position of L-tryptophan, in the first committed step of ergot alkaloid biosynthesis. It was previously shown to employ a dissociative mechanism, and two important catalytic residues, E89 and K174, have been identified from crystallographic studies. In this work, four mutants were prepared by mutating E89 and K174 to either glutamine or alanine. The results from kinetic studies and positional isotope exchange (PIX) experiments on all four mutants were consistent with the roles proposed for these two residues. Upon examination of the products in the mutant-catalyzed reactions, one unusual product was identified from the mutant K174A. A hexahydropyrroloindole structure was first proposed and later confirmed by obtaining an authentic sample through chemical synthesis. After examining the positioning of the substrates in the active site, a new mechanism involving a Cope rearrangement was proposed for DMATS. Another indole prenyltransferase CymD catalyzes a ‘reverse’ prenylation on the N-1 position of L-tryptophan. In this work, a series of mechanistic studies were carried out to probe its mechanism. Fluorinated tryptophan analogs demonstrated a modest effect on the rate of catalysis, suggesting no positive charge accumulation on the indole ring. A krel value of 1.0 × 10-² was determined with E-F-DMAPP, indicating that significant positive charge accumulates on the allylic moiety during the transition state of catalysis. PIX experiments with L-tryptophan did not show any isotopic scrambling, however, isotopic scrambling was observed with fluorinated tryptophans. This indicates that a discrete allylic carbocation intermediate is generated. Lastly, solvent kinetic studies presented a primary KIE of 2.3, indicating that the deprotonation of the N-H is a rate-determining step. A hybrid mechanism was proposed for CymD, in which dissociation first forms an allylic cation and then deprotonation direct the indole for nucleophilic attack.

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Inhibition Studies of the Detyrosination/Tyrosination Cycle (2014)

Microtubules are a highly dynamic component of the cytoskeleton, which are crucial for many cellular processes. Microtubules are comprised of α/β-tubulin heterodimers, which are subject to multiple post-translational modifications including the detyrosination/tyrosination cycle. This cycle involves the removal of an RNA-encoded C-terminal α-tubulin tyrosine residue by tubulin carboxypeptidase, followed by the reattachment of the tyrosine residue by tubulin tyrosine ligase. This research project is focused on the development of an inhibitor against tubulin tyrosine ligase and tubulin carboxypeptidase. The precise function of this cycle has yet to be determined; an inhibitor could function as a chemical biology tool that could be used to study the physiological effects of the detyrosination/tyrosination cycle. This thesis details the design and synthesis of phosphinic acid and phosphonic acid peptide analogue inhibitors. Progress towards the synthesis of a dipeptide phosphinic acid is reported; due to complications in phosphorus-carbon bond forming reactions the total synthesis was not completed. The focus of the research project changed to the synthesis of phosphonic acid peptide analogue inhibitors. A dipeptide phosphonic acid inhibitor was successfully synthesized, but was inactive against tubulin tyrosine ligase. Progress towards the synthesis of a tripeptide phosphonic acid inhibitor is reported; due to complications in the final deprotection steps the total synthesis was not completed. A pentapeptide phosphonic acid inhibitor was successfully synthesized, and it showed moderate inhibitor activity against tubulin tyrosine ligase.

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Peptidoglycan-modifying enzymes : mechanistic studies and substrate and inhibitor design (2011)

Peptidoglycan is a key component of the bacterial cell wall and is an essential structure for protecting the cell from lysis due to high osmotic pressure. Because of its importance, peptidoglycan has become a prominent target for antibiotic design as well as a number of host defense mechanisms. In response, many bacteria have developed methods of evading or minimizing the effects of these antibiotics and defense mechanisms through the modification of their peptidoglycan. One such modification, found in a number of bacterial species, is the O-acetylation of N-acetylmuramic acid (MurNAc) residues of peptidoglycan. This modification decreases the hydrolytic activity of lysozyme, an enzyme that is released as a response to bacterial infection, on peptidoglycan, and results in increased bacterial pathogenicity and virulence. The enzyme O-acetylpeptidoglycan esterase (Ape1) from Neisseria gonorrhoeae is an important enzyme involved in the bacterial O-acetylation/deacetylation pathway, and has been shown to be essential for bacterial viability. In this thesis, we detail the design and testing of water-soluble monosaccharide and disaccharide substrates of Ape1 (compounds 1 and 2) and preliminary work towards the design and testing of small-molecule inhibitors of the enzyme. Disaccharide 1 and monosaccharide 2 both served as substrates of Ape1, indicating that a polymeric substrate is not required for efficient catalysis. Our monosaccharide scaffold was chosen for the design of future generations of inhibitors. The enzyme N-acetylmuramic acid 6-phosphate hydrolase (MurQ) is essential for the recycling of MurNAc residues in peptidoglycan. This recycling process serves to reincorporate cell wall components into synthetic precursors that can be used in peptidoglycan biosynthesis as well as other basic metabolic pathways. MurQ catalyzes the conversion of MurNAc 6-phosphate to GlcNAc 6-phosphate through cleavage of a lactyl ether. In this thesis, studies on the mechanism through which MurQ catalyzes hydrolysis of the lactyl ether of MurNAc 6-phosphate are reported. By probing the reaction with chemically synthesized substrate and substrate analogues, an E1cB-like mechanism with an (E)-alkene intermediate is proposed. The amino acids Glu83 and Glu114 are implicated as important residues in catalysis, and their specific roles are also explored in the context of our proposed mechanism.

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Mechanistic Studies and Manipulation of the Enzymes in Sialic Acid and Pseudaminic Acid Biosynthesis (2010)

The Neisseria meningitidis sialic acid synthase (NeuB) catalyzes the metal-dependent condensation of N-acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP) to generate N-acetylneuraminic acid. This work describes the synthesis and characterization of the first potent inhibitor of sialic acid synthase, a tetrahedral intermediate analogue as a mixture of stereoisomers at the key tetrahedral center. Inhibition studies demonstrate that one stereoisomer binds more tightly than the other. An X-ray crystallographic analysis of the NeuB•inhibitor•Mn²⁺ complex solved to a resolution of 1.75 Å shows that the more tightly bound stereoisomer bears a (2R)-configuration. This suggests that the tetrahedral intermediate formed in the NeuB reaction also bears a (2R)-configuration. This analysis demonstrates that the active site metal serves as a source of nucleophilic water and delivers it to the si face of the oxocarbenium intermediate to generate a tetrahedral intermediate with a (2R)-configuration.The flagellin proteins in pathogenic bacteria such as Campylobacter jejuni and Helicobacter pylori are heavily glycosylated with the nine-carbon α-keto acid, pseudaminic acid. A key step in pseudaminic acid biosynthesis has been shown to involve the generation of 6-deoxy-AltdiNAc from its nucleotide-linked form, UDP-6-deoxy-AltdiNAc, by the action of a hydrolase that cleaves the glycosidic bond and releases UDP. This thesis describes the first characterization of a UDP-6-deoxy-AltdiNAc hydrolase, namely PseG (Cj1312) from C. jejuni. The activity of this enzyme is independent of the presence of divalent metal ions, and the values of the catalytic constants were found to be kcat = 27 s⁻­­­­­¹ and KM = 174 µM. The enzyme was shown to hydrolyze the substrate with an overall inversion of stereochemistry at C1 and to utilize a C-O bond cleavage mechanism during catalysis. The last part of the thesis describes the engineering of C. jejuni. We demonstrated that by feeding non-motile mutant C. jejuni bacteria with a neutral azide-labeled pseudaminic acid precursor, the mutants regained the ability to generate functional azido-bearing flagella and their motility was restored. The presence of the azido-pseudaminic acid on the surface of the flagella provides a bioorthogonal chemical handle that can be used to modify the flagellar proteins and to engineer bacteria for further studies.

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Mechanistic studies on (s)-norcoclaurine synthase and dimethylallyltryptophan synthase (2010)

In alkaloid biosynthesis, there are a limited number of enzymes that can catalyze an aromatic electrophilic substitution. One example is norcoclaurine synthase, which catalyzes an asymmetric Pictet-Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde to give (S)-norcoclaurine. This is the first committed step in the biosynthesis of the benzylisoquinoline alkaloids that include morphine and codeine. In this work, the gene encoding for the Thalictrum flavum norcoclaurine synthase is highly overexpressed in Escherichia coli and the His-tagged recombinant enzyme is purified for the first time. A continuous assay based on circular dichroism spectroscopy is developed and used to monitor the kinetics of the enzymatic reaction. Dopamine analogues bearing a methoxy or hydrogen substituent in place of the C-1 phenolic group were readily accepted by the enzyme whereas those bearing the same substituents at C-2 were not. This supports a mechanism involving a two-step cyclization of the putative iminium ion intermediate that does not proceed via a spirocyclic intermediate. The reaction of [3,5,6-²H₃]-dopamine was found to be slowed by a kinetic isotope effect of 1.7 ± 0.2 on the value of kcat/KM. This is interpreted as showing that the deprotonation step causing re-aromatization is partially rate determining in the overall reaction.Dimethylallyltryptophan synthase is an aromatic prenyltransferase that catalyzes an electrophilic aromatic substitution between dimethylallyl diphosphate (DMAPP) and L-tryptophan. The synthase catalyzes the first committed step in the ergot alkaloid biosynthesis. The enzymatic reaction could follow either an SN1 reaction involving a discrete dimethylallyl cation intermediate or an SN2 mechanism in which the indole ring directly displaces diphosphate in a single step. In this work, positional isotope exchange experiments are presented in support of an SN1 pathway. When [1-¹⁸O]-DMAPP is subjected to the synthase reaction, 15% of the ¹⁸O-label is found to have scrambled from a bridging to a non-bridging position on the α-phosphorus. Kinetic isotope effect studies show that steps involved in the formation of the arenium ion intermediate are rate-determining, and therefore the scrambling occurs during the lifetime of the dimethylallyl cation/diphosphate ion pair. Similarly, when the unreactive substrate analogue, 6-fluorotryptophan, was employed, complete scrambling of the ¹⁸O-label in DMAPP was observed.

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Mechanistic Studies on the Enzymes Involved in the Biosynthesis of CMP-N, N'Diacetyllegionaminic Acid and UDP-D-Apiose (2009)

No abstract available.

Master's Student Supervision (2010 - 2018)
Synthesis of peptidoglycan peptides for DNA aptamer selection (2018)

Gram-positive bacteria possess a thick layer of peptidoglycan outside the cell membrane that is rigidified through crosslinks between neighboring peptide chains on the polysaccharide structure.Vancomycin, a glycopeptide antibiotic, is effective at inhibiting the growth of Gram-positive bacteria by preventing the formation of crosslinks. The molecular basis of vancomycin’s action is tight binding to a cell wall peptide precursor that terminates in D-Ala-D-Ala. In vancomycin resistant bacteria, the D-Ala-D-Ala linkage is replaced by D-Ala-D-Lac and the loss of a hydrogen bond to the amide NH accounts for a 1000-fold loss in potency.In this research, we hope to generate DNA aptamers that will serve as an alternative to vancomycin and bind tightly to the peptidoglycan peptides. For DNA aptamer selection, two target compounds are synthesized. One molecule (Target 2) mimics the cell wall peptide precursor of vancomycin sensitive bacteria that terminates in L-Lys-D-Ala-D-Ala. Another molecule (Target 3) mimics the cell wall peptide precursor of vancomycin-resistant bacteria that terminates in L-Lys-D-Ala-D-Lac. This molecule will also be used to find catalytic unnatural DNA sequences (bearing primary amine groups) that can trans-amidate and displace the terminal lactate moiety. This will serve to form an agent that inactivates peptidoglycan biosynthesis of vancomycin resistant bacteria by forming a covalent linkage.

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Progress towards the synthesis of the bacterial menaquinone biosynthetic enzyme, 1,4-dihydroxy-6-naphthoate synthase (2014)

1,4-dihydroxy-6-naphthoate (DHN) synthase is one of several enzymes of the menaquinone biosynthetic pathway found in pathogenic bacteria including Campylobacter jejuni and Helicobacter pylori. It is responsible for catalyzing the formation of the 1,4-dihydroxy-6-naphthoate core of the electron transport system cofactor, menaquinone. Since humans lack the ability to synthesize menaquinone, enzymes of bacterial menaquinone biosynthesis have been targeted for inhibitor design. In order to aid the design of potential inhibitors of DHN synthase, its mechanism of catalysis has to be conclusively proven. We have proposed two possible reaction mechanisms that can be distinguished from one another, as they release different 3-carbon byproducts in addition to DHN. In order to establish the identity of these byproducts, and thus establish DHN synthase’s mechanism of action, hundred milligram quantities of substrate, much higher than the natural quantity of enzymatically-produced substrate, have to be synthesized. Accesss to synthetic substrate will allow for kinetic testing of potential inhibitors.In this thesis, progress towards the synthesis of two compounds will be presented. One is the natural substrate, CDHF, cyclic de-hypoxanthine futalosine and the other, de-carboxy CDHF, is a substrate analog devoid of a carboxylic acid functionality. We have demonstrated that an oxidative aromatic cyclization of a naphthol core successfully produces a key intermediate in the overall synthesis. This should ultimately allow for the completion of the synthesis of the substrate and the elucidation of the mechanism.

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