Angad Mehta - Academia.edu (original) (raw)

Papers by Angad Mehta

ACS Synthetic Biology

Essential viral enzymes have been successfully targeted to combat the diseases caused by emerging... more Essential viral enzymes have been successfully targeted to combat the diseases caused by emerging pathogenic RNA viruses (e.g., viral RNA-dependent RNA polymerase). Because of the conserved nature of such viral enzymes, therapeutics targeting these enzymes have the potential to be repurposed to combat emerging diseases, e.g., remdesivir, which was initially developed as a potential Ebola treatment, then was repurposed for COVID-19. Our efforts described in this study target another essential and highly conserved, but relatively less explored, step in RNA virus translation and replication, i.e., capping of the viral RNA genome. The viral genome cap structure disguises the genome of most RNA viruses to resemble the mRNA cap structure of their host and is essential for viral translation, propagation, and immune evasion. Here, we developed a synthetic, phenotypic yeast-based complementation platform (YeRC0M) for molecular characterization and targeting of SARS-CoV-2 genome-encoded RNA cap-0 (guanine-N7)-methyltransferase (N7-MTase) enzyme (nsp14). In YeRC0M, the lack of yeast mRNA capping N7-MTase in yeast, which is an essential gene in yeast, is complemented by the expression of functional viral N7-MTase or its variants. Using YeRC0M, we first identified important protein domains and amino acid residues that are essential for SARS-CoV-2 nsp14 N7-MTase activity. We also expanded YeRC0M to include key nsp14 variants observed in emerging variants of SARS-CoV-2 (e.g., delta variant of SARS-CoV-2 encodes nsp14 A394V and nsp14 P46L). We also combined YeRC0M with directed evolution to identify attenuation mutations in SARS-CoV-2 nsp14. Because of the high sequence similarity of nsp14 in emerging coronaviruses, these observations could have implications on live attenuated vaccine development strategies. These data taken together reveal key domains in SARS-CoV-2 nsp14 that can be targeted for therapeutic strategies. We also anticipate that these readily tractable phenotypic platforms can also be used for the identification of inhibitors of viral RNA capping enzymes as antivirals.

Nature Communications

The evolutionary origin of the photosynthetic eukaryotes drastically altered the evolution of com... more The evolutionary origin of the photosynthetic eukaryotes drastically altered the evolution of complex lifeforms and impacted global ecology. The endosymbiotic theory suggests that photosynthetic eukaryotes evolved due to endosymbiosis between non-photosynthetic eukaryotic host cells and photosynthetic cyanobacterial or algal endosymbionts. The photosynthetic endosymbionts, propagating within the cytoplasm of the host cells, evolved, and eventually transformed into chloroplasts. Despite the fundamental importance of this evolutionary event, we have minimal understanding of this remarkable evolutionary transformation. Here, we design and engineer artificial, genetically tractable, photosynthetic endosymbiosis between photosynthetic cyanobacteria and budding yeasts. We engineer various mutants of model photosynthetic cyanobacteria as endosymbionts within yeast cells where, the engineered cyanobacteria perform bioenergetic functions to support the growth of yeast cells under defined pho...

This dissertation focuses on radical S-adenosylmethionine enzymes involved in cofactor biosynthes... more This dissertation focuses on radical S-adenosylmethionine enzymes involved in cofactor biosynthesis. Mechanistic studies discussed here include: (i) molybdenum cofactor biosynthetic enzyme-MoaA, (ii) thiamin pyrimidine synthase-ThiC (iii) hydroxybenzimidazole synthase, HBI synthase, involved in anaerobic vitamin B 12 biosynthesis. MoaA catalyzes the first step in molydopterin biosynthesis where GTP is converted to pterin. This catalysis involves a remarkable rearrangement reaction where the C8 of guanosine-5'-triphosphate (GTP) is inserted between the C2' and C3' carbon atoms of GTP to give the final pterin. Mechanistic studies involved characterization of the products of the reaction, identification of the position of hydrogen atom abstraction by 5'-deoxyadenosyl radical and trapping of intermediates by using 2',3'-dideoxyGTP, 2'-deoxyGTP and 2'-chloroGTP as substrate analogs. Thiamin pyrimidine synthase, ThiC, catalyzes a complex rearrangement reaction involving the conversion of aminoimidazole ribotide (AIR) to thiamin pyrimidine (HMP-P). A hydrogen atom transfer from S-adenosylmethionine (AdoMet) to HMP-P was demonstrated. Also, the stereochemistry of this transfer was elucidated. Bioinformatics studies on ThiC revealed that a paralog of ThiC was clustered with vitamin B 12 biosynthetic genes in several anaerobic microorganisms. The gene responsible for the anaerobic vitamin B 12-benzimidazole biosynthesis was previously unknown. We demonstrate that the gene product of this ThiC paralog is a radical Siii adenosylmethionine enzyme. Remarkably it catalyzes the conversion of aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (5-HBI) and formate, and Sadenosylmethionine to 5'-deoxyadenosine. We determine the hydrogen atom abstracted by 5'-deoxyadenosyl radical. We also performed carbon, nitrogen and hydrogen labeling studies and characterized the labeling pattern on 5-HBI. Based on these studies we propose a reaction mechanism of this remarkable conversion of AIR to 5-HBI.

Journal of the American Chemical Society, 2018

Organisms that perform the de novo biosynthesis of cobalamin (vitamin B12) do so via unique pathw... more Organisms that perform the de novo biosynthesis of cobalamin (vitamin B12) do so via unique pathways depending on the presence of oxygen in the environment. The anaerobic biosynthesis pathway of 5,6-dimethylbenzimidazole, the so-called "lower ligand" to the cobalt center, has been recently identified. This process begins with the conversion of 5-aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (HBI) by the radical S-adenosyl-L-methionine (SAM) enzyme BzaF, also known as HBI synthase. In this work we report the characterization of a radical intermediate in the reaction of BzaF using electron paramagnetic resonance (EPR) spectroscopy. Using various isotopologues of AIR, we extracted hyperfine parameters for a number of nuclei, allowing us to propose plausible chemical compositions and structures for this intermediate. Specifically, we find that an aminoimidazole radical is formed in close proximity to a fragment of the ribose ring. These findings induce the revision of past proposed mechanisms and illustrate the ability of radical SAM enzymes to tightly control the radical chemistry that they engender.

Journal of the American Chemical Society, 2019

Based on the endosymbiotic theory, one of the key events that occurred during mitochondrial evolu... more Based on the endosymbiotic theory, one of the key events that occurred during mitochondrial evolution was an extensive loss of non-essential genes from the protomitochondrial endosymbiont genome and transfer of some of the essential endosymbiont genes to the host nucleus. We have developed an approach to recapitulate various aspects of endosymbiont genome minimization using a synthetic system consisting of E. coli endosymbionts within host yeast cells. As a first step, we identified a number of E. coli auxotrophs of central metabolites that can form viable endosymbionts within yeast cells. These studies provide a platform to identify non-essential biosynthetic pathways that can be deleted in the E. coli endosymbionts to investigate the evolutionary adaptations in the host and endosymbiont during the evolution of mitochondria.

Journal of the American Chemical Society, 2018

The ability to add noncanonical amino acids to the genetic code may allow one to evolve proteins ... more The ability to add noncanonical amino acids to the genetic code may allow one to evolve proteins with new or enhanced properties using a larger set of building blocks. To this end, we have been able to select mutant proteins with enhanced thermal properties from a library of E. coli homoserine o-succinyltransferase (metA) mutants containing randomly incorporated noncanonical amino acids. Here, we show that substitution of Phe 21 with p-benzoylphenylalanine (pBzF), increases the melting temperature of E. coli metA by 21°C. This dramatic increase in thermal stability, arising from a single mutation, likely results from a covalent adduct between Cys 90 and the keto group of pBzF that stabilizes the dimeric form of the enzyme. These experiments show that an expanded genetic code can provide unique solutions to the evolution of proteins with enhanced properties.

Proceedings of the National Academy of Sciences, 2018

Significance Endosymbiotic theory suggests that mitochondria evolved from free-living prokaryotes... more Significance Endosymbiotic theory suggests that mitochondria evolved from free-living prokaryotes which entered the host cell and were retained as endosymbionts. Here, we model this earliest stage of the endosymbiotic theory of mitochondrial evolution by engineering endosymbiosis between two genetically tractable model organisms, Escherichia coli and Saccharomyces cerevisiae . In this model system, we engineered E. coli strains to survive in the yeast cytosol and provide ATP to a respiration-deficient yeast mutant. In a reciprocal fashion, yeast provided thiamin to an endosymbiotic E. coli thiamin auxotroph. This readily manipulated chimeric system was stable for more than 40 doublings and should allow us to investigate various aspects of the endosymbiotic theory of mitochondrial evolution.

Journal of the American Chemical Society, Jan 12, 2018

Almost five decades ago Crick, Orgel, and others proposed the RNA world hypothesis. Subsequent st... more Almost five decades ago Crick, Orgel, and others proposed the RNA world hypothesis. Subsequent studies have raised the possibility that RNA might be able to support both genotype and phenotype, and the function of RNA templates has been studied in terms of evolution, replication, and catalysis. Recently, we engineered strains of E. coli in which a large fraction of 2'-deoxycytidine in the genome is substituted with the modified base 5-hydroxymethyl-2'-deoxycytidine. We now report the generation of mutant strains derived from these engineered bacteria that show significant (∼40-50%) ribonucleotide content in their genome. We have begun to characterize the properties of these chimeric genomes and the corresponding strains to determine the circumstances under which E. coli can incorporate ribonucleotides into its genome and herein report our initial observations.

Bioorganic & medicinal chemistry, Jan 26, 2018

All known living organisms use at least 20 amino acids as the basic building blocks of life. Effo... more All known living organisms use at least 20 amino acids as the basic building blocks of life. Efforts to reduce the number of building blocks in a replicating system to below the 20 canonical amino acids have not been successful to date. In this work, we use filamentous phage as a model system to investigate the feasibility of removing methionine (Met) from the proteome. We show that all 24 elongation Met sites in the M13 phage genome can be replaced by other canonical amino acids. Most of these changes involve substitution of methionine by leucine (Leu), but in some cases additional compensatory mutations are required. Combining Met substituted sites in the proteome generally led to lower viability/infectivity of the mutant phages, which remains the major challenge in eliminating all methionines from the phage proteome. To date a total of 15 (out of all 24) elongation Mets have been simultaneously deleted from the M13 proteome, providing a useful foundation for future efforts to min...

Journal of the American Chemical Society, 2017

Materials: E. coli DH10B was used for general molecular biology purposes. LB and TB medium were p... more Materials: E. coli DH10B was used for general molecular biology purposes. LB and TB medium were purchased from Corning, LB agar purchased from Fisher Scientific, Isopropyl-ß-thiogalactoside (IPTG) was purchased from Anatrace, 4-12% (wt/vol) Bis-Tris gels for SDS-PAGE were purchased from Invitrogen, Q5 DNA Site-Directed Mutagenesis Kit, restriction enzymes and T4 DNA ligase were obtained from New England Biolabs (NEB) and oligonucleotides were purchased from Integrated DNA Technologies (San Diego, CA). Plasmid DNA preparation was carried out with QIAprep Spin Miniprep Kit (Quiagen). Unless otherwise mentioned, all chemicals and solvents were purchased from Sigma-Aldrich and used without further purification. Peptide concentrations were measured using BCA assay from Pierce. LCMS conditions for the analysis of Nisin analogs: Agilent 6520 accurate-mass quadrupole-time-of-flight (QTOF) instrument was used to carry out high-resolution mass spectrometry for all peptide samples, which was equipped with reverse phase liquid chromatography and an electrospray ionization (ESI) source. Samples in distilled deionized water were injected (10-15 µL) at a concentration of 0.1 mg/mL and separated on a 150 mm reverse phase C8 wide pore column heated to 70 °C to improve peak resolution (Phenomenex, Aeris WIDEPORE XBC8, LC column 150 x 2.1 mm). Peptides were eluted in a gradient of H 2 O + 0.1% formic acid (solvent A) and acetonitrile + 0.1% formic acid (solvent B) using the following method: 5% B for 2 min, 5-60% B for 10 min, 60-80% B for 1 min, followed by a wash (95% B) and re-equilibration (5% A) phase. ESI source settings were 350 °C, 10 L/min, 40 psig nebulizer nitrogen gas, 200 V fragmentor and 4,500 V capillary. Figures were created by extraction of the total ion count and deconvolution of charge envelopes using Agilent Qualitative Analysis software with BioConfirm. NMR for Nisin analogs: 1 H/ 13 C NMR spectra were recorded at ambient temperature on Bruker DMX 400 (400 MHz for 1 H NMR and 100 MHz for 13 C NMR) and DRX 600 CryoProbe (600 MHz for 1 H NMR and 150 MHz for 13 C NMR) instruments in the specified deuterated solvents. Chemicals shifts are given in ppm with respect to residual undeuterated solvent signal as internal standard. Coupling constants are reported as J-values in Hz. The following abbreviations were used to explain S2 multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet, m = multiplet, br = broad. Chemical synthesis 1 H and 13 C-NMR spectrum of 13 C-labelled (1): Chloroacetamide-containing ncAA and its 13 C-labelled version were prepared as follows: Boc-4-Amino-L-Phenylalnine (purchased by Ark chemicals) (2g, 7.1 mmol, 1 eq) and NaHCO 3 (3g, 35 mmol, 5 eq) were suspended in anhydrous THF (50 mL) and brought to 0 °C using an icewater bath. Subsequently, chloroacetyl chloride or chloroacetyl chloride-2-13 C for isotope labeling experiment (3.5 mL, 43.74 mmol, 6.25 eq) in anhydrous THF (17.5 mL) was added dropwise, and the reaction stirred overnight shielded from light using aluminum wrap. LC-MS analysis showed full conversion to the desired product, at this point, 4% NaHCO 3 (aq) (10 mL) was added, and the mixture extracted with EtOAc (30 mL 3X). Organic layers were pooled, dried over Na 2 SO 4 , filtered and dried in vacuo. The residue was then taken up in CH 2 Cl 2 (15 mL), brought to 0 °C and then treated with anhydrous TFA (3 mL). Upon complete Boc deprotection, as observed by LC-MS, the reaction mixture was dried in vacuo, and the residue taken up in 1:1 H 2 O-CH 3 CN and freeze-dried to obtain a white powder in 70-85% yield. Products were used without any further purification. 13 C-labelled chloroacetamide 1, was prepared as described previously but using chloroacetyl chloride-2-13 C, purchased from Sigma-Aldrich (CAS# 604097).

Journal of Biological Chemistry, 2016

Riboflavin is a common cofactor, and its biosynthetic pathway is well characterized. However, its... more Riboflavin is a common cofactor, and its biosynthetic pathway is well characterized. However, its catabolic pathway, despite intriguing hints in a few distinct organisms, has never been established. This article describes the isolation of a Microbacterium maritypicum riboflavin catabolic strain, and the cloning of the riboflavin catabolic genes. RcaA, RcaB, RcaD, and RcaE were overexpressed and biochemically characterized as riboflavin kinase, riboflavin reductase, ribokinase, and riboflavin hydrolase, respectively. Based on these activities, a pathway for riboflavin catabolism is proposed.

Journal of the American Chemical Society, Nov 20, 2016

Several modified bases have been observed in the genomic DNA of bacteriophages, prokaryotes, and ... more Several modified bases have been observed in the genomic DNA of bacteriophages, prokaryotes, and eukaryotes that play a role in restriction systems and/or epigenetic regulation. In our efforts to understand the consequences of replacing a large fraction of a canonical nucleoside with a modified nucleoside, we previously replaced around 75% of thymidine (T) with 5'-hydroxymethyl-2'-deoxyuridine (5hmU) in the Escherichia coli genome. In this study, we engineered the pyrimidine nucleotide biosynthetic pathway using T4 bacteriophage genes to achieve approximately 63% replacement of 2'-deoxycytidine (dC) with 5-hydroxymethyl-2'-deoxycytidine (5hmC) in the E. coli genome and approximately 71% replacement in plasmids. We further engineered the glucose metabolic pathway to transform the 5hmC into glucosyl-5-hydroxymethyl-2'-deoxycytidine (5-gmC) and achieved 20% 5-gmC in the genome and 45% 5-gmC in plasmid DNA.

Journal of the American Chemical Society, Jun 23, 2016

Prokaryotic and eukaryotic genomic DNA is comprised of the four building blocks A, G, C and T. We... more Prokaryotic and eukaryotic genomic DNA is comprised of the four building blocks A, G, C and T. We have be-gun to explore the consequences of replacing a large fraction or all of a nucleoside in genomic DNA with a modified nucleoside. As a first step we have investigated the possibility of replacement of T by 2'-deoxy-5-(hydroxymethyl)uridine (5hmU) in the genomic DNA of E. coli. Metabolic engineering with phage genes fol-lowed by random mutagenesis enabled us to achieve approximately 75% replacement of T by 5hmU in the E. coli genome and in plasmids.

Proceedings of the National Academy of Sciences of the United States of America, Jan 25, 2015

Vitamin B12 (cobalamin) is required by humans and other organisms for diverse metabolic processes... more Vitamin B12 (cobalamin) is required by humans and other organisms for diverse metabolic processes, although only a subset of prokaryotes is capable of synthesizing B12 and other cobamide cofactors. The complete aerobic and anaerobic pathways for the de novo biosynthesis of B12 are known, with the exception of the steps leading to the anaerobic biosynthesis of the lower ligand, 5,6-dimethylbenzimidazole (DMB). Here, we report the identification and characterization of the complete pathway for anaerobic DMB biosynthesis. This pathway, identified in the obligate anaerobic bacterium Eubacterium limosum, is composed of five previously uncharacterized genes, bzaABCDE, that together direct DMB production when expressed in anaerobically cultured Escherichia coli. Expression of different combinations of the bza genes revealed that 5-hydroxybenzimidazole, 5-methoxybenzimidazole, and 5-methoxy-6-methylbenzimidazole, all of which are lower ligands of cobamides produced by other organisms, are i...

Journal of the American Chemical Society, Jan 3, 2015

Comparative genomics of the bacterial thiamin pyrimidine synthase (thiC) revealed a paralog of th... more Comparative genomics of the bacterial thiamin pyrimidine synthase (thiC) revealed a paralog of thiC (thiC-1) clustered with anaerobic vitamin B12 biosynthetic genes. Here we demonstrate that ThiC-1 is a radical S-adenosylmethionine enzyme that catalyzes the remarkable conversion of aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (5-HBI). We identify the origin of key product atoms and propose a reaction mechanism. These studies represent the first step in solving a long-standing problem in anaerobic vitamin B12 assembly and reveal an unanticipated intersection of thiamin and vitamin B12 biosynthesis.

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2015

ACS Synthetic Biology

Essential viral enzymes have been successfully targeted to combat the diseases caused by emerging... more Essential viral enzymes have been successfully targeted to combat the diseases caused by emerging pathogenic RNA viruses (e.g., viral RNA-dependent RNA polymerase). Because of the conserved nature of such viral enzymes, therapeutics targeting these enzymes have the potential to be repurposed to combat emerging diseases, e.g., remdesivir, which was initially developed as a potential Ebola treatment, then was repurposed for COVID-19. Our efforts described in this study target another essential and highly conserved, but relatively less explored, step in RNA virus translation and replication, i.e., capping of the viral RNA genome. The viral genome cap structure disguises the genome of most RNA viruses to resemble the mRNA cap structure of their host and is essential for viral translation, propagation, and immune evasion. Here, we developed a synthetic, phenotypic yeast-based complementation platform (YeRC0M) for molecular characterization and targeting of SARS-CoV-2 genome-encoded RNA cap-0 (guanine-N7)-methyltransferase (N7-MTase) enzyme (nsp14). In YeRC0M, the lack of yeast mRNA capping N7-MTase in yeast, which is an essential gene in yeast, is complemented by the expression of functional viral N7-MTase or its variants. Using YeRC0M, we first identified important protein domains and amino acid residues that are essential for SARS-CoV-2 nsp14 N7-MTase activity. We also expanded YeRC0M to include key nsp14 variants observed in emerging variants of SARS-CoV-2 (e.g., delta variant of SARS-CoV-2 encodes nsp14 A394V and nsp14 P46L). We also combined YeRC0M with directed evolution to identify attenuation mutations in SARS-CoV-2 nsp14. Because of the high sequence similarity of nsp14 in emerging coronaviruses, these observations could have implications on live attenuated vaccine development strategies. These data taken together reveal key domains in SARS-CoV-2 nsp14 that can be targeted for therapeutic strategies. We also anticipate that these readily tractable phenotypic platforms can also be used for the identification of inhibitors of viral RNA capping enzymes as antivirals.

Nature Communications

The evolutionary origin of the photosynthetic eukaryotes drastically altered the evolution of com... more The evolutionary origin of the photosynthetic eukaryotes drastically altered the evolution of complex lifeforms and impacted global ecology. The endosymbiotic theory suggests that photosynthetic eukaryotes evolved due to endosymbiosis between non-photosynthetic eukaryotic host cells and photosynthetic cyanobacterial or algal endosymbionts. The photosynthetic endosymbionts, propagating within the cytoplasm of the host cells, evolved, and eventually transformed into chloroplasts. Despite the fundamental importance of this evolutionary event, we have minimal understanding of this remarkable evolutionary transformation. Here, we design and engineer artificial, genetically tractable, photosynthetic endosymbiosis between photosynthetic cyanobacteria and budding yeasts. We engineer various mutants of model photosynthetic cyanobacteria as endosymbionts within yeast cells where, the engineered cyanobacteria perform bioenergetic functions to support the growth of yeast cells under defined pho...

This dissertation focuses on radical S-adenosylmethionine enzymes involved in cofactor biosynthes... more This dissertation focuses on radical S-adenosylmethionine enzymes involved in cofactor biosynthesis. Mechanistic studies discussed here include: (i) molybdenum cofactor biosynthetic enzyme-MoaA, (ii) thiamin pyrimidine synthase-ThiC (iii) hydroxybenzimidazole synthase, HBI synthase, involved in anaerobic vitamin B 12 biosynthesis. MoaA catalyzes the first step in molydopterin biosynthesis where GTP is converted to pterin. This catalysis involves a remarkable rearrangement reaction where the C8 of guanosine-5'-triphosphate (GTP) is inserted between the C2' and C3' carbon atoms of GTP to give the final pterin. Mechanistic studies involved characterization of the products of the reaction, identification of the position of hydrogen atom abstraction by 5'-deoxyadenosyl radical and trapping of intermediates by using 2',3'-dideoxyGTP, 2'-deoxyGTP and 2'-chloroGTP as substrate analogs. Thiamin pyrimidine synthase, ThiC, catalyzes a complex rearrangement reaction involving the conversion of aminoimidazole ribotide (AIR) to thiamin pyrimidine (HMP-P). A hydrogen atom transfer from S-adenosylmethionine (AdoMet) to HMP-P was demonstrated. Also, the stereochemistry of this transfer was elucidated. Bioinformatics studies on ThiC revealed that a paralog of ThiC was clustered with vitamin B 12 biosynthetic genes in several anaerobic microorganisms. The gene responsible for the anaerobic vitamin B 12-benzimidazole biosynthesis was previously unknown. We demonstrate that the gene product of this ThiC paralog is a radical Siii adenosylmethionine enzyme. Remarkably it catalyzes the conversion of aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (5-HBI) and formate, and Sadenosylmethionine to 5'-deoxyadenosine. We determine the hydrogen atom abstracted by 5'-deoxyadenosyl radical. We also performed carbon, nitrogen and hydrogen labeling studies and characterized the labeling pattern on 5-HBI. Based on these studies we propose a reaction mechanism of this remarkable conversion of AIR to 5-HBI.

Journal of the American Chemical Society, 2018

Organisms that perform the de novo biosynthesis of cobalamin (vitamin B12) do so via unique pathw... more Organisms that perform the de novo biosynthesis of cobalamin (vitamin B12) do so via unique pathways depending on the presence of oxygen in the environment. The anaerobic biosynthesis pathway of 5,6-dimethylbenzimidazole, the so-called "lower ligand" to the cobalt center, has been recently identified. This process begins with the conversion of 5-aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (HBI) by the radical S-adenosyl-L-methionine (SAM) enzyme BzaF, also known as HBI synthase. In this work we report the characterization of a radical intermediate in the reaction of BzaF using electron paramagnetic resonance (EPR) spectroscopy. Using various isotopologues of AIR, we extracted hyperfine parameters for a number of nuclei, allowing us to propose plausible chemical compositions and structures for this intermediate. Specifically, we find that an aminoimidazole radical is formed in close proximity to a fragment of the ribose ring. These findings induce the revision of past proposed mechanisms and illustrate the ability of radical SAM enzymes to tightly control the radical chemistry that they engender.

Journal of the American Chemical Society, 2019

Based on the endosymbiotic theory, one of the key events that occurred during mitochondrial evolu... more Based on the endosymbiotic theory, one of the key events that occurred during mitochondrial evolution was an extensive loss of non-essential genes from the protomitochondrial endosymbiont genome and transfer of some of the essential endosymbiont genes to the host nucleus. We have developed an approach to recapitulate various aspects of endosymbiont genome minimization using a synthetic system consisting of E. coli endosymbionts within host yeast cells. As a first step, we identified a number of E. coli auxotrophs of central metabolites that can form viable endosymbionts within yeast cells. These studies provide a platform to identify non-essential biosynthetic pathways that can be deleted in the E. coli endosymbionts to investigate the evolutionary adaptations in the host and endosymbiont during the evolution of mitochondria.

Journal of the American Chemical Society, 2018

The ability to add noncanonical amino acids to the genetic code may allow one to evolve proteins ... more The ability to add noncanonical amino acids to the genetic code may allow one to evolve proteins with new or enhanced properties using a larger set of building blocks. To this end, we have been able to select mutant proteins with enhanced thermal properties from a library of E. coli homoserine o-succinyltransferase (metA) mutants containing randomly incorporated noncanonical amino acids. Here, we show that substitution of Phe 21 with p-benzoylphenylalanine (pBzF), increases the melting temperature of E. coli metA by 21°C. This dramatic increase in thermal stability, arising from a single mutation, likely results from a covalent adduct between Cys 90 and the keto group of pBzF that stabilizes the dimeric form of the enzyme. These experiments show that an expanded genetic code can provide unique solutions to the evolution of proteins with enhanced properties.

Proceedings of the National Academy of Sciences, 2018

Significance Endosymbiotic theory suggests that mitochondria evolved from free-living prokaryotes... more Significance Endosymbiotic theory suggests that mitochondria evolved from free-living prokaryotes which entered the host cell and were retained as endosymbionts. Here, we model this earliest stage of the endosymbiotic theory of mitochondrial evolution by engineering endosymbiosis between two genetically tractable model organisms, Escherichia coli and Saccharomyces cerevisiae . In this model system, we engineered E. coli strains to survive in the yeast cytosol and provide ATP to a respiration-deficient yeast mutant. In a reciprocal fashion, yeast provided thiamin to an endosymbiotic E. coli thiamin auxotroph. This readily manipulated chimeric system was stable for more than 40 doublings and should allow us to investigate various aspects of the endosymbiotic theory of mitochondrial evolution.

Journal of the American Chemical Society, Jan 12, 2018

Almost five decades ago Crick, Orgel, and others proposed the RNA world hypothesis. Subsequent st... more Almost five decades ago Crick, Orgel, and others proposed the RNA world hypothesis. Subsequent studies have raised the possibility that RNA might be able to support both genotype and phenotype, and the function of RNA templates has been studied in terms of evolution, replication, and catalysis. Recently, we engineered strains of E. coli in which a large fraction of 2'-deoxycytidine in the genome is substituted with the modified base 5-hydroxymethyl-2'-deoxycytidine. We now report the generation of mutant strains derived from these engineered bacteria that show significant (∼40-50%) ribonucleotide content in their genome. We have begun to characterize the properties of these chimeric genomes and the corresponding strains to determine the circumstances under which E. coli can incorporate ribonucleotides into its genome and herein report our initial observations.

Bioorganic & medicinal chemistry, Jan 26, 2018

All known living organisms use at least 20 amino acids as the basic building blocks of life. Effo... more All known living organisms use at least 20 amino acids as the basic building blocks of life. Efforts to reduce the number of building blocks in a replicating system to below the 20 canonical amino acids have not been successful to date. In this work, we use filamentous phage as a model system to investigate the feasibility of removing methionine (Met) from the proteome. We show that all 24 elongation Met sites in the M13 phage genome can be replaced by other canonical amino acids. Most of these changes involve substitution of methionine by leucine (Leu), but in some cases additional compensatory mutations are required. Combining Met substituted sites in the proteome generally led to lower viability/infectivity of the mutant phages, which remains the major challenge in eliminating all methionines from the phage proteome. To date a total of 15 (out of all 24) elongation Mets have been simultaneously deleted from the M13 proteome, providing a useful foundation for future efforts to min...

Journal of the American Chemical Society, 2017

Materials: E. coli DH10B was used for general molecular biology purposes. LB and TB medium were p... more Materials: E. coli DH10B was used for general molecular biology purposes. LB and TB medium were purchased from Corning, LB agar purchased from Fisher Scientific, Isopropyl-ß-thiogalactoside (IPTG) was purchased from Anatrace, 4-12% (wt/vol) Bis-Tris gels for SDS-PAGE were purchased from Invitrogen, Q5 DNA Site-Directed Mutagenesis Kit, restriction enzymes and T4 DNA ligase were obtained from New England Biolabs (NEB) and oligonucleotides were purchased from Integrated DNA Technologies (San Diego, CA). Plasmid DNA preparation was carried out with QIAprep Spin Miniprep Kit (Quiagen). Unless otherwise mentioned, all chemicals and solvents were purchased from Sigma-Aldrich and used without further purification. Peptide concentrations were measured using BCA assay from Pierce. LCMS conditions for the analysis of Nisin analogs: Agilent 6520 accurate-mass quadrupole-time-of-flight (QTOF) instrument was used to carry out high-resolution mass spectrometry for all peptide samples, which was equipped with reverse phase liquid chromatography and an electrospray ionization (ESI) source. Samples in distilled deionized water were injected (10-15 µL) at a concentration of 0.1 mg/mL and separated on a 150 mm reverse phase C8 wide pore column heated to 70 °C to improve peak resolution (Phenomenex, Aeris WIDEPORE XBC8, LC column 150 x 2.1 mm). Peptides were eluted in a gradient of H 2 O + 0.1% formic acid (solvent A) and acetonitrile + 0.1% formic acid (solvent B) using the following method: 5% B for 2 min, 5-60% B for 10 min, 60-80% B for 1 min, followed by a wash (95% B) and re-equilibration (5% A) phase. ESI source settings were 350 °C, 10 L/min, 40 psig nebulizer nitrogen gas, 200 V fragmentor and 4,500 V capillary. Figures were created by extraction of the total ion count and deconvolution of charge envelopes using Agilent Qualitative Analysis software with BioConfirm. NMR for Nisin analogs: 1 H/ 13 C NMR spectra were recorded at ambient temperature on Bruker DMX 400 (400 MHz for 1 H NMR and 100 MHz for 13 C NMR) and DRX 600 CryoProbe (600 MHz for 1 H NMR and 150 MHz for 13 C NMR) instruments in the specified deuterated solvents. Chemicals shifts are given in ppm with respect to residual undeuterated solvent signal as internal standard. Coupling constants are reported as J-values in Hz. The following abbreviations were used to explain S2 multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet, m = multiplet, br = broad. Chemical synthesis 1 H and 13 C-NMR spectrum of 13 C-labelled (1): Chloroacetamide-containing ncAA and its 13 C-labelled version were prepared as follows: Boc-4-Amino-L-Phenylalnine (purchased by Ark chemicals) (2g, 7.1 mmol, 1 eq) and NaHCO 3 (3g, 35 mmol, 5 eq) were suspended in anhydrous THF (50 mL) and brought to 0 °C using an icewater bath. Subsequently, chloroacetyl chloride or chloroacetyl chloride-2-13 C for isotope labeling experiment (3.5 mL, 43.74 mmol, 6.25 eq) in anhydrous THF (17.5 mL) was added dropwise, and the reaction stirred overnight shielded from light using aluminum wrap. LC-MS analysis showed full conversion to the desired product, at this point, 4% NaHCO 3 (aq) (10 mL) was added, and the mixture extracted with EtOAc (30 mL 3X). Organic layers were pooled, dried over Na 2 SO 4 , filtered and dried in vacuo. The residue was then taken up in CH 2 Cl 2 (15 mL), brought to 0 °C and then treated with anhydrous TFA (3 mL). Upon complete Boc deprotection, as observed by LC-MS, the reaction mixture was dried in vacuo, and the residue taken up in 1:1 H 2 O-CH 3 CN and freeze-dried to obtain a white powder in 70-85% yield. Products were used without any further purification. 13 C-labelled chloroacetamide 1, was prepared as described previously but using chloroacetyl chloride-2-13 C, purchased from Sigma-Aldrich (CAS# 604097).

Journal of Biological Chemistry, 2016

Riboflavin is a common cofactor, and its biosynthetic pathway is well characterized. However, its... more Riboflavin is a common cofactor, and its biosynthetic pathway is well characterized. However, its catabolic pathway, despite intriguing hints in a few distinct organisms, has never been established. This article describes the isolation of a Microbacterium maritypicum riboflavin catabolic strain, and the cloning of the riboflavin catabolic genes. RcaA, RcaB, RcaD, and RcaE were overexpressed and biochemically characterized as riboflavin kinase, riboflavin reductase, ribokinase, and riboflavin hydrolase, respectively. Based on these activities, a pathway for riboflavin catabolism is proposed.

Journal of the American Chemical Society, Nov 20, 2016

Several modified bases have been observed in the genomic DNA of bacteriophages, prokaryotes, and ... more Several modified bases have been observed in the genomic DNA of bacteriophages, prokaryotes, and eukaryotes that play a role in restriction systems and/or epigenetic regulation. In our efforts to understand the consequences of replacing a large fraction of a canonical nucleoside with a modified nucleoside, we previously replaced around 75% of thymidine (T) with 5'-hydroxymethyl-2'-deoxyuridine (5hmU) in the Escherichia coli genome. In this study, we engineered the pyrimidine nucleotide biosynthetic pathway using T4 bacteriophage genes to achieve approximately 63% replacement of 2'-deoxycytidine (dC) with 5-hydroxymethyl-2'-deoxycytidine (5hmC) in the E. coli genome and approximately 71% replacement in plasmids. We further engineered the glucose metabolic pathway to transform the 5hmC into glucosyl-5-hydroxymethyl-2'-deoxycytidine (5-gmC) and achieved 20% 5-gmC in the genome and 45% 5-gmC in plasmid DNA.

Journal of the American Chemical Society, Jun 23, 2016

Prokaryotic and eukaryotic genomic DNA is comprised of the four building blocks A, G, C and T. We... more Prokaryotic and eukaryotic genomic DNA is comprised of the four building blocks A, G, C and T. We have be-gun to explore the consequences of replacing a large fraction or all of a nucleoside in genomic DNA with a modified nucleoside. As a first step we have investigated the possibility of replacement of T by 2'-deoxy-5-(hydroxymethyl)uridine (5hmU) in the genomic DNA of E. coli. Metabolic engineering with phage genes fol-lowed by random mutagenesis enabled us to achieve approximately 75% replacement of T by 5hmU in the E. coli genome and in plasmids.

Proceedings of the National Academy of Sciences of the United States of America, Jan 25, 2015

Vitamin B12 (cobalamin) is required by humans and other organisms for diverse metabolic processes... more Vitamin B12 (cobalamin) is required by humans and other organisms for diverse metabolic processes, although only a subset of prokaryotes is capable of synthesizing B12 and other cobamide cofactors. The complete aerobic and anaerobic pathways for the de novo biosynthesis of B12 are known, with the exception of the steps leading to the anaerobic biosynthesis of the lower ligand, 5,6-dimethylbenzimidazole (DMB). Here, we report the identification and characterization of the complete pathway for anaerobic DMB biosynthesis. This pathway, identified in the obligate anaerobic bacterium Eubacterium limosum, is composed of five previously uncharacterized genes, bzaABCDE, that together direct DMB production when expressed in anaerobically cultured Escherichia coli. Expression of different combinations of the bza genes revealed that 5-hydroxybenzimidazole, 5-methoxybenzimidazole, and 5-methoxy-6-methylbenzimidazole, all of which are lower ligands of cobamides produced by other organisms, are i...

Journal of the American Chemical Society, Jan 3, 2015

Comparative genomics of the bacterial thiamin pyrimidine synthase (thiC) revealed a paralog of th... more Comparative genomics of the bacterial thiamin pyrimidine synthase (thiC) revealed a paralog of thiC (thiC-1) clustered with anaerobic vitamin B12 biosynthetic genes. Here we demonstrate that ThiC-1 is a radical S-adenosylmethionine enzyme that catalyzes the remarkable conversion of aminoimidazole ribotide (AIR) to 5-hydroxybenzimidazole (5-HBI). We identify the origin of key product atoms and propose a reaction mechanism. These studies represent the first step in solving a long-standing problem in anaerobic vitamin B12 assembly and reveal an unanticipated intersection of thiamin and vitamin B12 biosynthesis.

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2015