Synthetic and Biochemical Exploration of the Degradation and Utilization of Thiamin Analogs and Preliminary Studies on Methanopterin methyltransferase

Thiaminase I from Clostridium botulinum cleaves thiamin to the constituent pyrimidine and thiazole using a wide range of external nucleophiles. The crystal structure of the thiamin bound mutant of thiaminase I from Clostridium botulinum revealed the complete active site architecture and all the catalytically important residues were identified. The role of each active site residue was determined by its position and the steady-state kinetic study of the mutant of the corresponding residue. Based on the structural and the kinetic data, the mechanism of thiaminase I is proposed. Thiaminase I accepts a wide variety of cysteine containing nucleophiles, suggesting the possibility of some protein or peptide as its natural nucleophile. A plate assay and an HPLC based assay were developed to identify new thiaminase producing bacteria. The second part of the thesis is focused on the development of synthetic strategies for thiamin analogs to answer the following biological questions. Firstly, methoxythiamin pyrophosphate synthesized to study the effect of bacimethrin on thiamin-dependent enzymes and ^13C and ^15N labeled thiamin analog was synthesized to study different intermediate states of thiamin in PDC. Inhibition of thiamin-dependent enzymes was observed and the data indicates that the toxicity arises due to a different binding mode of 2’-methoxythiamin in the active site. Secondly, two fluorine labeled thiamin analogs were designed that can be used as a tracer for PET imaging study in live animals. Thirdly, synthetic methodologies have been developed to utilize thiamin molecule as a delivery vehicle. The impermeable cargo molecules attached to thiamin molecule via ester or carbamate linkage can be delivered inside the cells through membrane transporters of thiamin. Finally, synthesis of thiochrome was utilized to estimate thiamin content in auxotrophic phytoplankton and preferential utilization of pyrimidine precursors were observed to fulfill the thiamin requirement. Preliminary studies were done to explore a putative methanopterin methyltransferase MJ0619. This enzyme is air sensitive and copurifies with bound 4Fe- 4S clusters. MJ0619 contains at least three 4Fe-4S clusters and can cleave SAM homolytically in reducing conditions, which classifies it as a radical SAM enzyme. It also possesses GTP cyclohydrolase activity to produce 7,8-dihydroneopterin cyclic phosphate from GTP and can cleave N-glycosidic bond of several nucleosides. Methylation of 7,8- dihydro-6-hydroxymethyl pterin is also observed with this enzyme, however the sources of the methyl groups are still unknown

Synthetic and Biochemical Exploration of the Degradation and Utilization of Thiamin Analogs and Preliminary Studies on Methanopterin methyltransferase

Thiaminase I from Clostridium botulinum cleaves thiamin to the constituent pyrimidine and thiazole using a wide range of external nucleophiles. The crystal structure of the thiamin bound mutant of thiaminase I from Clostridium botulinum revealed the complete active site architecture and all the catalytically important residues were identified. The role of each active site residue was determined by its position and the steady-state kinetic study of the mutant of the corresponding residue. Based on the structural and the kinetic data, the mechanism of thiaminase I is proposed. Thiaminase I accepts a wide variety of cysteine containing nucleophiles, suggesting the possibility of some protein or peptide as its natural nucleophile. A plate assay and an HPLC based assay were developed to identify new thiaminase producing bacteria. The second part of the thesis is focused on the development of synthetic strategies for thiamin analogs to answer the following biological questions. Firstly, methoxythiamin pyrophosphate synthesized to study the effect of bacimethrin on thiamin-dependent enzymes and ^13C and ^15N labeled thiamin analog was synthesized to study different intermediate states of thiamin in PDC. Inhibition of thiamin-dependent enzymes was observed and the data indicates that the toxicity arises due to a different binding mode of 2’-methoxythiamin in the active site. Secondly, two fluorine labeled thiamin analogs were designed that can be used as a tracer for PET imaging study in live animals. Thirdly, synthetic methodologies have been developed to utilize thiamin molecule as a delivery vehicle. The impermeable cargo molecules attached to thiamin molecule via ester or carbamate linkage can be delivered inside the cells through membrane transporters of thiamin. Finally, synthesis of thiochrome was utilized to estimate thiamin content in auxotrophic phytoplankton and preferential utilization of pyrimidine precursors were observed to fulfill the thiamin requirement. Preliminary studies were done to explore a putative methanopterin methyltransferase MJ0619. This enzyme is air sensitive and copurifies with bound 4Fe- 4S clusters. MJ0619 contains at least three 4Fe-4S clusters and can cleave SAM homolytically in reducing conditions, which classifies it as a radical SAM enzyme. It also possesses GTP cyclohydrolase activity to produce 7,8-dihydroneopterin cyclic phosphate from GTP and can cleave N-glycosidic bond of several nucleosides. Methylation of 7,8- dihydro-6-hydroxymethyl pterin is also observed with this enzyme, however the sources of the methyl groups are still unknown

Synthetic and Biochemical Exploration of the Degradation and Utilization of Thiamin Analogs and Preliminary Studies on Methanopterin methyltransferase

Thiaminase I from Clostridium botulinum cleaves thiamin to the constituent pyrimidine and thiazole using a wide range of external nucleophiles. The crystal structure of the thiamin bound mutant of thiaminase I from Clostridium botulinum revealed the complete active site architecture and all the catalytically important residues were identified. The role of each active site residue was determined by its position and the steady-state kinetic study of the mutant of the corresponding residue. Based on the structural and the kinetic data, the mechanism of thiaminase I is proposed. Thiaminase I accepts a wide variety of cysteine containing nucleophiles, suggesting the possibility of some protein or peptide as its natural nucleophile. A plate assay and an HPLC based assay were developed to identify new thiaminase producing bacteria. The second part of the thesis is focused on the development of synthetic strategies for thiamin analogs to answer the following biological questions. Firstly, methoxythiamin pyrophosphate synthesized to study the effect of bacimethrin on thiamin-dependent enzymes and ^13C and ^15N labeled thiamin analog was synthesized to study different intermediate states of thiamin in PDC. Inhibition of thiamin-dependent enzymes was observed and the data indicates that the toxicity arises due to a different binding mode of 2’-methoxythiamin in the active site. Secondly, two fluorine labeled thiamin analogs were designed that can be used as a tracer for PET imaging study in live animals. Thirdly, synthetic methodologies have been developed to utilize thiamin molecule as a delivery vehicle. The impermeable cargo molecules attached to thiamin molecule via ester or carbamate linkage can be delivered inside the cells through membrane transporters of thiamin. Finally, synthesis of thiochrome was utilized to estimate thiamin content in auxotrophic phytoplankton and preferential utilization of pyrimidine precursors were observed to fulfill the thiamin requirement. Preliminary studies were done to explore a putative methanopterin methyltransferase MJ0619. This enzyme is air sensitive and copurifies with bound 4Fe- 4S clusters. MJ0619 contains at least three 4Fe-4S clusters and can cleave SAM homolytically in reducing conditions, which classifies it as a radical SAM enzyme. It also possesses GTP cyclohydrolase activity to produce 7,8-dihydroneopterin cyclic phosphate from GTP and can cleave N-glycosidic bond of several nucleosides. Methylation of 7,8- dihydro-6-hydroxymethyl pterin is also observed with this enzyme, however the sources of the methyl groups are still unknown

Globally important haptophyte algae use exogenous pyrimidine compounds more efficiently than thiamin

Vitamin B1 (thiamin) is a cofactor for critical enzymatic processes and is scarce in surface oceans. Several eukaryotic marine algal species thought to rely on exogenous thiamin are now known to grow equally well on the precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), including the haptophyte Emiliania huxleyi. Because the thiamin biosynthetic capacities of the diverse and ecologically important haptophyte lineage are otherwise unknown, we investigated the pathway in transcriptomes and two genomes from 30 species representing six taxonomic orders. HMP synthase is missing in data from all studied taxa, but the pathway is otherwise complete, with some enzymatic variations. Experiments on axenic species from three orders demonstrated that equivalent growth rates were supported by 1 μM HMP or thiamin amendment. Cellular thiamin quotas were quantified in the oceanic phytoplankter E. huxleyi using the thiochrome assay. E. huxleyi exhibited luxury storage in standard algal medium (1.16 ± 0.18) ☓ 10-6 pmol thiamin cell-1, whereas quotas in cultures grown under more environmentally relevant thiamin and HMP supplies (2.22 ± 0.07) ☓ 10-7 or (1.58 ± 0.14) ☓ 10-7 pmol thiamin cell-1, respectively were significantly lower than luxury values and prior estimates. HMP and its salvage-related analog 4-amino-5-aminomethyl-2-methylpyrimidine (AmMP) supported higher growth than thiamin under environmentally relevant supply levels. These compounds also sustained growth of the stramenopile alga Pelago-monas calceolata. Together with identification of a salvage protein subfamily (TENA_E) in multiple phytoplankton, the results indicate that salvaged AmMP and exogenously acquired HMP are used by several groups for thiamin production. Our studies highlight the potential importance of thiamin pathway intermediates and their analogs in shaping phytoplankton community structure. IMPORTANCE The concept that vitamin B1 (thiamin) availability in seawater controls the productivity and structure of eukaryotic phytoplankton communities has been discussed for half a century. We examined B1 biosynthesis and salvage pathways in diverse phytoplankton species. These comparative genomic analyses as well as experiments show that phytoplankton thought to require exogenous B1 not only utilize intermediate compounds to meet this need but also exhibit stronger growth on these compounds than on thiamin. Furthermore, oceanic phytoplankton have lower cellular thiamin quotas than previously reported, and salvage of intermediate compounds is likely a key mechanism for meeting B1 requirements under environmentally relevant scenarios. Thus, several lines of evidence now suggest that availability of specific precursor molecules could be more important in structuring phytoplankton communities than the vitamin itself. This understanding of preferential compound utilization and thiamin quotas will improve biogeochemical model pa-rameterization and highlights interaction networks among ocean microbes. © 2017 Gutowska et al

Structure of a <i>Clostridium botulinum</i> C143S Thiaminase I/Thiamin Complex Reveals Active Site Architecture,

Thiaminases are responsible for the degradation of thiamin and its metabolites. Two classes of thiaminases have been identified based on their three-dimensional structures and their requirements for a nucleophilic second substrate. Although the reactions of several thiaminases have been characterized, the physiological role of thiamin degradation is not fully understood. We have determined the three-dimensional X-ray structure of an inactive C143S mutant of Clostridium botulinum (Cb) thiaminase I with bound thiamin at 2.2 Å resolution. The C143S/thiamin complex provides atomic level details of the orientation of thiamin upon binding to Cb-thiaminase I and the identity of active site residues involved in substrate binding and catalysis. The specific roles of active site residues were probed by using site directed mutagenesis and kinetic analyses, leading to a detailed mechanism for Cb-thiaminase I. The structure of Cb-thiaminase I is also compared to the functionally similar but structurally distinct thiaminase II

Competence of Thiamin Diphosphate-Dependent Enzymes with 2′-Methoxythiamin Diphosphate Derived from Bacimethrin, a Naturally Occurring Thiamin Anti-vitamin

Bacimethrin (4-amino-5-hydroxymethyl-2-methoxypyrimidine), a natural product isolated from some bacteria, has been implicated as an inhibitor of bacterial and yeast growth, as well as in inhibition of thiamin biosynthesis. Given that thiamin biosynthetic enzymes could convert bacimethrin to 2′-methoxythiamin diphosphate (MeOThDP), it is important to evaluate the effect of this coenzyme analogue on thiamin diphosphate (ThDP)-dependent enzymes. The potential functions of MeOThDP were explored on five ThDP-dependent enzymes: the human and <i>Escherichia coli</i> pyruvate dehydrogenase complexes (PDHc-h and PDHc-ec, respectively), the <i>E. coli</i> 1-deoxy-d-xylulose 5-phosphate synthase (DXPS), and the human and <i>E. coli</i> 2-oxoglutarate dehydrogenase complexes (OGDHc-h and OGDHc-ec, respectively). Using several mechanistic tools (fluorescence, circular dichroism, kinetics, and mass spectrometry), it was demonstrated that MeOThDP binds in the active centers of ThDP-dependent enzymes, however, with a binding mode different from that of ThDP. While modest activities resulted from addition of MeOThDP to <i>E. coli</i> PDHc (6–11%) and DXPS (9–14%), suggesting that MeOThDP-derived covalent intermediates are converted to the corresponding products (albeit with rates slower than that with ThDP), remarkably strong activity (up to 75%) resulted upon addition of the coenzyme analogue to PDHc-h. With PDHc-ec and PDHc-h, the coenzyme analogue could support all reactions, including communication between components in the complex. No functional substitution of MeOThDP for ThDP was in evidence with either OGDH-h or OGDH-ec, shown to be due to tight binding of ThDP

Structural basis for antibiotic action of the B1 antivitamin 2′-methoxy-thiamine

The natural antivitamin 2′-methoxy-thiamine (MTh) is implicated in the suppression of microbial growth. However, its mode of action and enzyme-selective inhibition mechanism have remained elusive. Intriguingly, MTh inhibits some thiamine diphosphate (ThDP) enzymes, while being coenzymatically active in others. Here we report the strong inhibition of Escherichia coli transketolase activity by MTh and unravel its mode of action and the structural basis thereof. The unique 2′-methoxy group of MTh diphosphate (MThDP) clashes with a canonical glutamate required for cofactor activation in ThDP-dependent enzymes. This glutamate is forced into a stable, anticatalytic low-barrier hydrogen bond with a neighboring glutamate, disrupting cofactor activation. Molecular dynamics simulations of transketolases and other ThDP enzymes identify active-site flexibility and the topology of the cofactor-binding locale as key determinants for enzyme-selective inhibition. Human enzymes either retain enzymatic activity with MThDP or preferentially bind authentic ThDP over MThDP, while core bacterial metabolic enzymes are inhibited, demonstrating therapeutic potential