Search for: All records

Exchanging the native iron of heme for other metals yields artificial metalloproteins with new properties for spectroscopic studies and biocatalysis. Recently, we reported a method for the biosynthesis and incorporation of a non-natural metallocofactor, cobalt protoporphyrin IX (CoPPIX), into hemoproteins using the common laboratory strain Escherichia coli BL21(DE3). This discovery inspired us to explore the determinants of metal specificity for metallocofactor biosynthesis in E. coli. Herein, we report detailed kinetic analysis of the ferrochelatase responsible for metal insertion, EcHemH (E. coli ferrochelatase). This enzyme exhibits a small, less than 2-fold preference for Fe2+ over the non-native Co2+ substrate in vitro. To test how mutations impact EcHemH, we used a surrogate metal specificity screen to identify variants with altered metal insertion preferences. This engineering process led to a variant with an ∼30-fold shift in specificity toward Co2+. When assayed in vivo, however, the impact of this mutation is small compared to the effects of alteration of the external metal concentrations. These data suggest that incorporation of cobalt into PPIX is enabled by the native promiscuity of EcHemH coupled with BL21’s impaired ability to maintain transition-metal homeostasis. With this knowledge, we generated a method for CoPPIX production in rich media, which yields cobalt-substituted hemoproteins with >95% cofactor purity and yields comparable to standard expression protocols for the analogous native hemoproteins. 

Non‐canonical amino acids (ncAAs) are useful synthons for the development of new medicines, materials, and probes for bioactivity. Recently, enzyme engineering has been leveraged to produce a suite of highly active enzymes for the synthesis of β‐substituted amino acids. However, there are few examples of biocatalyticN‐substitution reactions to make α,β‐diamino acids. In this study, we used directed evolution to engineer the β‐subunit of tryptophan synthase, TrpB, for improved activity with diverse amine nucleophiles. Mechanistic analysis shows that high yields are hindered by product re‐entry into the catalytic cycle and subsequent decomposition. Additional equivalents ofl‐serine can inhibit product reentry through kinetic competition, facilitating preparative scale synthesis. We show β‐substitution with a dozen aryl amine nucleophiles, including demonstration on a gram scale. These transformations yield an underexplored class of amino acids that can serve as unique building blocks for chemical biology and medicinal chemistry.

 

Non‐canonical amino acids (ncAAs) are useful synthons for the development of new medicines, materials, and probes for bioactivity. Recently, enzyme engineering has been leveraged to produce a suite of highly active enzymes for the synthesis of β‐substituted amino acids. However, there are few examples of biocatalyticN‐substitution reactions to make α,β‐diamino acids. In this study, we used directed evolution to engineer the β‐subunit of tryptophan synthase, TrpB, for improved activity with diverse amine nucleophiles. Mechanistic analysis shows that high yields are hindered by product re‐entry into the catalytic cycle and subsequent decomposition. Additional equivalents ofl‐serine can inhibit product reentry through kinetic competition, facilitating preparative scale synthesis. We show β‐substitution with a dozen aryl amine nucleophiles, including demonstration on a gram scale. These transformations yield an underexplored class of amino acids that can serve as unique building blocks for chemical biology and medicinal chemistry.

 

Enzymes with high activity are readily produced through protein engineering, but intentionally and efficiently engineering enzymes for an expanded substrate scope is a contemporary challenge. One approach to address this challenge is Substrate Multiplexed Screening (SUMS), where enzyme activity is measured on competing substrates. SUMS has long been used to rigorously quantitate native enzyme specificity, primarily for in vivo settings. SUMS has more recently found sporadic use as a protein engineering approach but has not been widely adopted by the field, despite its potential utility. Here, we develop principles of how to design and interpret SUMS assays to guide protein engineering. This rich information enables improving activity with multiple substrates simultaneously, identifies enzyme variants with altered scope, and indicates potential mutational hot-spots as sites for further engineering. These advances leverage common laboratory equipment and represent a highly accessible and customizable method for enzyme engineering.

 

Biocatalytic cascades are uniquely powerful for the efficient, asymmetric synthesis of bioactive compounds. However, high substrate specificity can hinder the scope of biocatalytic cascades because the constituent enzymes may have non‐complementary activity. In this study, we implemented a substrate multiplexed screening (SUMS) based directed evolution approach to improve the substrate scope overlap between a transaldolase (ObiH) and a decarboxylase for the production of chiral 1,2‐amino alcohols. To generate a promiscuous cascade, we engineered a tryptophan decarboxylase to act efficiently on β‐OH amino acids while avoiding activity onl‐threonine, which is needed for ObiH activity. We leveraged this exquisite selectivity with matched substrate scope to produce a variety of enantiopure 1,2‐amino alcohols in a one‐pot cascade from aldehydes or styrene oxides. This demonstration shows how SUMS can be used to guide the development of promiscuous, C−C bond forming cascades.

 

Biocatalytic cascades are uniquely powerful for the efficient, asymmetric synthesis of bioactive compounds. However, high substrate specificity can hinder the scope of biocatalytic cascades because the constituent enzymes may have non‐complementary activity. In this study, we implemented a substrate multiplexed screening (SUMS) based directed evolution approach to improve the substrate scope overlap between a transaldolase (ObiH) and a decarboxylase for the production of chiral 1,2‐amino alcohols. To generate a promiscuous cascade, we engineered a tryptophan decarboxylase to act efficiently on β‐OH amino acids while avoiding activity onl‐threonine, which is needed for ObiH activity. We leveraged this exquisite selectivity with matched substrate scope to produce a variety of enantiopure 1,2‐amino alcohols in a one‐pot cascade from aldehydes or styrene oxides. This demonstration shows how SUMS can be used to guide the development of promiscuous, C−C bond forming cascades.

 

Enzymes from secondary metabolic pathways possess broad potential for the selective synthesis of complex bioactive molecules. However, the practical application of these enzymes for organic synthesis is dependent on the development of efficient, economical, operationally simple, and well‐characterized systems for preparative scale reactions. We sought to bridge this knowledge gap for the selective biocatalytic synthesis of β‐hydroxy‐α‐amino acids, which are important synthetic building blocks. To achieve this goal, we demonstrated the ability of ObiH, anl‐threonine transaldolase, to achieve selective milligram‐scale synthesis of a diverse array of non‐standard amino acids (nsAAs) using a scalable whole cell platform. We show how the initial selectivity of the catalyst is high and how the diastereomeric ratio of products decreases at high conversion due to product re‐entry into the catalytic cycle. ObiH‐catalyzed reactions with a variety of aromatic, aliphatic and heterocyclic aldehydes selectively generated a panel of β‐hydroxy‐α‐amino acids possessing broad functional‐group diversity. Furthermore, we demonstrated that ObiH‐generated β‐hydroxy‐α‐amino acids could be modified through additional transformations to access important motifs, such as β‐chloro‐α‐amino acids and substituted α‐keto acids.