Engineering bioorthogonal reactions on proteins in live cells

This project deals with a method to optimize in vivo labeling using small fluorescent molecules via bioorthogonal reactions. The reaction used involves our unnatural amino acid 4-(6-methyl-s-tetrazin-3-yl)aminophenylalanine (AMT-Phe). The amino acid is site-specifically incorporated into GFP and then reacted with a labeled, strained trans-cyclooctene, resulting in a labeled protein. However we have noticed that the label becomes detached from the protein. The conditions and influential factors that cause this are unknown, so tests to determine the stability of AMT-Phe were run using mass spectrometry. Results have shown that AMT-Phe might be decomposing once incorporated in the protein over time, suggesting that it’s not very stable. AMT-Phe contains tetrazine functionality, with a methyl group attached to the tetrazine. A potentially more stable version of AMT-Phe is to replace the methyl group with a phenyl group. This potentially more stable and reactive amino acid, 4-(6-methyl-s-tetrazin-3-yl)aminophenyl- alanine (APT-Phe), was synthesized, and a series of tests on incorporation and reactive rates were run to compare with AMT-Phe. Although APT-Phe didn’t incorporate into GFP very well, preliminary data shows that it has a 6-fold faster reaction rate than AMT-Phe

Engineering bioorthogonal reactions on proteins in live cells

This project deals with a method to optimize in vivo labeling using small fluorescent molecules via bioorthogonal reactions. The reaction used involves our unnatural amino acid 4-(6-methyl-s-tetrazin-3-yl)aminophenylalanine (AMT-Phe). The amino acid is site-specifically incorporated into GFP and then reacted with a labeled, strained trans-cyclooctene, resulting in a labeled protein. However we have noticed that the label becomes detached from the protein. The conditions and influential factors that cause this are unknown, so tests to determine the stability of AMT-Phe were run using mass spectrometry. Results have shown that AMT-Phe might be decomposing once incorporated in the protein over time, suggesting that it’s not very stable. AMT-Phe contains tetrazine functionality, with a methyl group attached to the tetrazine. A potentially more stable version of AMT-Phe is to replace the methyl group with a phenyl group. This potentially more stable and reactive amino acid, 4-(6-methyl-s-tetrazin-3-yl)aminophenyl- alanine (APT-Phe), was synthesized, and a series of tests on incorporation and reactive rates were run to compare with AMT-Phe. Although APT-Phe didn’t incorporate into GFP very well, preliminary data shows that it has a 6-fold faster reaction rate than AMT-Phe

A promiscuous cytochrome P450 aromatic O-demethylase for lignin bioconversion

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOMicrobial aromatic catabolism offers a promising approach to convert lignin, a vast source of renewable carbon, into useful products. Aryl-O-demethylation is an essential biochemical reaction to ultimately catabolize coniferyl and sinapyl lignin-derived a9FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO2013/08293-72014/10448-12016/22956-7We acknowledge funding from NSF grants to J.L.D. (MCB-1715176), K.N.H. (CHE-1361104), and E.L.N. (DEB-1556541 and MCB-1615365) and BBSRC grants to J.E.M. (BB/P011918/1, BB/L001926/1 and a studentship to S.J.B.M.). G.T.B., M.M.M., C.W.J., M.F.C., E.L.N.,

Enabling microbial syringol conversion through structure-guided protein engineering

Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds

Machovina CUE poster.pdf

This project deals with a method to optimize in vivo labeling using small fluorescent molecules via bioorthogonal reactions. The reaction used involves our unnatural amino acid 4-(6-methyl-s-tetrazin-3-yl)aminophenylalanine (AMT-Phe). The amino acid is site-specifically incorporated into GFP and then reacted with a labeled, strained trans-cyclooctene, resulting in a labeled protein. However we have noticed that the label becomes detached from the protein. The conditions and influential factors that cause this are unknown, so tests to determine the stability of AMT-Phe were run using mass spectrometry. Results have shown that AMT-Phe might be decomposing once incorporated in the protein over time, suggesting that it’s not very stable. AMT-Phe contains tetrazine functionality, with a methyl group attached to the tetrazine. A potentially more stable version of AMT-Phe is to replace the methyl group with a phenyl group. This potentially more stable and reactive amino acid, 4-(6-methyl-s-tetrazin-3-yl)aminophenyl- alanine (APT-Phe), was synthesized, and a series of tests on incorporation and reactive rates were run to compare with AMT-Phe. Although APT-Phe didn’t incorporate into GFP very well, preliminary data shows that it has a 6-fold faster reaction rate than AMT-Phe.Keywords: unnatural amino acids, BioorthogonalKeywords: unnatural amino acids, Bioorthogona

Machovina CUE poster.pptx

This project deals with a method to optimize in vivo labeling using small fluorescent molecules via bioorthogonal reactions. The reaction used involves our unnatural amino acid 4-(6-methyl-s-tetrazin-3-yl)aminophenylalanine (AMT-Phe). The amino acid is site-specifically incorporated into GFP and then reacted with a labeled, strained trans-cyclooctene, resulting in a labeled protein. However we have noticed that the label becomes detached from the protein. The conditions and influential factors that cause this are unknown, so tests to determine the stability of AMT-Phe were run using mass spectrometry. Results have shown that AMT-Phe might be decomposing once incorporated in the protein over time, suggesting that it’s not very stable. AMT-Phe contains tetrazine functionality, with a methyl group attached to the tetrazine. A potentially more stable version of AMT-Phe is to replace the methyl group with a phenyl group. This potentially more stable and reactive amino acid, 4-(6-methyl-s-tetrazin-3-yl)aminophenyl- alanine (APT-Phe), was synthesized, and a series of tests on incorporation and reactive rates were run to compare with AMT-Phe. Although APT-Phe didn’t incorporate into GFP very well, preliminary data shows that it has a 6-fold faster reaction rate than AMT-Phe.Keywords: unnatural amino acids, Bioorthogona

Machovina CUE poster.pptx

This project deals with a method to optimize in vivo labeling using small fluorescent molecules via bioorthogonal reactions. The reaction used involves our unnatural amino acid 4-(6-methyl-s-tetrazin-3-yl)aminophenylalanine (AMT-Phe). The amino acid is site-specifically incorporated into GFP and then reacted with a labeled, strained trans-cyclooctene, resulting in a labeled protein. However we have noticed that the label becomes detached from the protein. The conditions and influential factors that cause this are unknown, so tests to determine the stability of AMT-Phe were run using mass spectrometry. Results have shown that AMT-Phe might be decomposing once incorporated in the protein over time, suggesting that it’s not very stable. AMT-Phe contains tetrazine functionality, with a methyl group attached to the tetrazine. A potentially more stable version of AMT-Phe is to replace the methyl group with a phenyl group. This potentially more stable and reactive amino acid, 4-(6-methyl-s-tetrazin-3-yl)aminophenyl- alanine (APT-Phe), was synthesized, and a series of tests on incorporation and reactive rates were run to compare with AMT-Phe. Although APT-Phe didn’t incorporate into GFP very well, preliminary data shows that it has a 6-fold faster reaction rate than AMT-Phe.Keywords: unnatural amino acids, Bioorthogona

A promiscuous cytochrome P450 aromatic O-demethylase for lignin bioconversion.

Microbial aromatic catabolism offers a promising approach to convert lignin, a vast source of renewable carbon, into useful products. Aryl-O-demethylation is an essential biochemical reaction to ultimately catabolize coniferyl and sinapyl lignin-derived aromatic compounds, and is often a key bottleneck for both native and engineered bioconversion pathways. Here, we report the comprehensive characterization of a promiscuous P450 aryl-O-demethylase, consisting of a cytochrome P450 protein from the family CYP255A (GcoA) and a three-domain reductase (GcoB) that together represent a new two-component P450 class. Though originally described as converting guaiacol to catechol, we show that this system efficiently demethylates both guaiacol and an unexpectedly wide variety of lignin-relevant monomers. Structural, biochemical, and computational studies of this novel two-component system elucidate the mechanism of its broad substrate specificity, presenting it as a new tool for a critical step in biological lignin conversion