Molecular Determinants of Efficient Cobalt-Substituted Hemoprotein Production in E. coli.

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.

Multiplexed Assessment of Promiscuous Non-Canonical Amino Acid Synthase Activity in a Pyridoxal Phosphate-Dependent Protein Family.

Pyridoxal phosphate (PLP)-dependent enzymes afford access to a variety of non-canonical amino acids (ncAAs), which are premier buildings blocks for the construction of complex bioactive molecules. The vinylglycine ketimine (VGK) subfamily of PLP-dependent enzymes plays a critical role in sulfur metabolism and is home to a growing set of secondary metabolic enzymes that synthesize γ-substituted ncAAs. Identification of VGK enzymes for biocatalysis faces a distinct challenge because the subfamily contains both desirable synthases as well as lyases that break down ncAAs. Some enzymes have both activities, which may contribute to pervasive mis-annotation. To navigate this complex functional landscape, we used a substrate multiplexed screening approach to rapidly measure the substrate promiscuity of 40 homologs in the VGK subfamily. We found that enzymes involved in transsulfuration are less likely to have promiscuous activities and often possess undesirable lyase activity. Enzymes from direct sulfuration and secondary metabolism generally had a high degree of substrate promiscuity. From this cohort, we identified an exemplary γ-synthase from Caldicellulosiruptor hydrothermalis (CahyGS). This enzyme is thermostable and has high expression (~400 mg protein per L culture), enabling preparative scale synthesis of thioether containing ncAAs. When assayed with l-allylglycine, CahyGS catalyzes a stereoselective γ-addition reaction to afford access to a unique set of γ-methyl branched ncAAs. We determined high-resolution crystal structures of this enzyme that define an open-close transition associated with ligand binding and set the stage for future engineering within this enzyme subfamily.

Engineered Biocatalytic Synthesis of ß-N-Substituted-α-Amino Acids.

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 ß-substituted amino acids. However, there are few examples of biocatalytic N-substitution reactions to make α,ß-diamino acids. In this study, we used directed evolution to engineer the ß-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 of l-serine can inhibit product reentry through kinetic competition, facilitating preparative scale synthesis. We show ß-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.

Efficient chemoenzymatic synthesis of α-aryl aldehydes as intermediates in C-C bond forming biocatalytic cascades.

Multi-enzyme biocatalytic cascades are emerging as practical routes for the synthesis of complex bioactive molecules. However, the relative sparsity of water-stable carbon electrophiles limits the synthetic complexity of molecules made from such cascades. Here, we develop a chemoenzymatic platform that leverages styrene oxide isomerase (SOI) to covert readily accessible aryl epoxides into α-aryl aldehydes through a Meinwald rearrangement. These unstable aldehyde intermediates are then intercepted with a C-C bond forming enzyme, ObiH, that catalyzes a transaldolase reaction with l-threonine to yield synthetically challenging ß-hydroxy-α-amino acids. Co-expression of both enzymes in E. coli yields a whole cell biocatalyst capable of synthesizing a variety of stereopure non-standard amino acids (nsAA) and can be produced on gram-scale. We used isotopically labelled substrates to probe the mechanism of SOI, which we show catalyzes a concerted isomerization featuring a stereospecific 1,2-hydride shift. The viability of in situ generated α-aryl aldehydes was further established by intercepting them with a recently engineered decarboxylative aldolase to yield γ-hydroxy nsAAs. Together, these data establish a versatile method of producing α-aryl aldehydes in simple, whole cell conditions and show that these intermediates are useful synthons in C‒C bond forming cascades.

Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis.

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.

Engineering Enzyme Substrate Scope Complementarity for Promiscuous Cascade Synthesis of 1,2-Amino Alcohols.

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 ß-OH amino acids while avoiding activity on l-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 synthesis of non-standard amino acids by a decarboxylative aldol reaction.

Enzymes are renowned for their catalytic efficiency and selectivity. Despite the wealth of carbon-carbon bond forming transformations in traditional organic chemistry and nature, relatively few C-C bond forming enzymes have found their way into the biocatalysis toolbox. Here we show that the enzyme UstD performs a highly selective decarboxylative aldol addition with diverse aldehyde substrates to make non-standard, γ-hydroxy amino acids. We increased the activity of UstD through three rounds of classic directed evolution and an additional round of computationally-guided engineering. The enzyme that emerged, UstDv2.0, is efficient in a whole-cell biocatalysis format. The products are highly desirable, functionally rich bioactive γ-hydroxy amino acids that we demonstrate can be prepared stereoselectively on gram-scale. The X-ray crystal structure of UstDv2.0 at 2.25 Šreveals the active site and provides a foundation for probing the mechanism of UstD.

Investigation of ß-Substitution Activity of O-Acetylserine Sulfhydrolase from Citrullus vulgaris.

Pyridoxal-5'-phosphate (PLP)-dependent enzymes have garnered interest for their ability to synthesize non-standard amino acids (nsAAs). One such class of enzymes, O-acetylserine sulfhydrylases (OASSs), catalyzes the final step in the biosynthesis of l-cysteine. Here, we examine the ß-substitution capability of the OASS from Citrullus vulgaris (CvOASS), a putative l-mimosine synthase. While the previously reported mimosine synthase activity was not reproducible in our hands, we successfully identified non-native reactivity with a variety of O-nucleophiles. Optimization of reaction conditions for carboxylate and phenolate substrates led to distinct conditions that were leveraged for the preparative-scale synthesis of nsAAs. We further show this enzyme is capable of C-C bond formation through a ß-alkylation reaction with an activated nitroalkane. To facilitate understanding of this enzyme, we determined the crystal structure of the enzyme bound to PLP as the internal aldimine at 1.55 Å, revealing key features of the active site and providing information that may guide subsequent development of CvOASS as a practical biocatalyst.

Site-Selective Deuteration of Amino Acids through Dual-Protein Catalysis.

Deuterated amino acids have been recognized for their utility in drug development, for facilitating nuclear magnetic resonance (NMR) analysis, and as probes for enzyme mechanism. Small molecule-based methods for the site-selective synthesis of deuterated amino acids typically involve de novo synthesis of the compound from deuterated precursors. In comparison, enzymatic methods for introducing deuterium offer improved efficiency, operating directly on free amino acids to achieve hydrogen-deuterium (H/D) exchange. However, site selectivity remains a significant challenge for enzyme-mediated deuteration, limiting access to desirable deuteration motifs. Here, we use enzyme-catalyzed deuteration, combined with steady-state kinetic analysis and ultraviolet (UV)-vis spectroscopy to probe the mechanism of a two-protein system responsible for the biosynthesis of l-allo-Ile. We show that an aminotransferase (DsaD) can pair with a small partner protein (DsaE) to catalyze Cα and Cß H/D exchange of amino acids, while reactions without DsaE lead exclusively to Cα-deuteration. With conditions for improved catalysis, we evaluate the substrate scope for Cα/Cß-deuteration and demonstrate the utility of this system for preparative-scale, selective labeling of amino acids.

Scalable and Selective ß-Hydroxy-α-Amino Acid Synthesis Catalyzed by Promiscuous l-Threonine Transaldolase ObiH.

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 ß-hydroxy-α-amino acids, which are important synthetic building blocks. To achieve this goal, we demonstrated the ability of ObiH, an l-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 ß-hydroxy-α-amino acids possessing broad functional-group diversity. Furthermore, we demonstrated that ObiH-generated ß-hydroxy-α-amino acids could be modified through additional transformations to access important motifs, such as ß-chloro-α-amino acids and substituted α-keto acids.

Asymmetric Alkylation of Ketones Catalyzed by Engineered TrpB.

The ß-subunit of tryptophan synthase (TrpB) catalyzes a PLP-mediated ß-substitution reaction between indole and serine to form L-Trp. A succession of TrpB protein engineering campaigns to expand the enzyme's nucleophile substrate range has enabled the biocatalytic production of diverse non-canonical amino acids (ncAAs). Here, we show that ketone-derived enolates can serve as nucleophiles in the TrpB reaction to achieve the asymmetric alkylation of ketones, an outstanding challenge in synthetic chemistry. We engineered TrpB by directed evolution to catalyze the asymmetric alkylation of propiophenone and 2-fluoroacetophenone with a high degree of selectivity. In reactions with propiophenone, preference for the opposite product diastereomer emerges over the course of evolution, demonstrating that full control over the stereochemistry at the new chiral center can be achieved. The addition of this new reaction to the TrpB platform is a crucial first step toward the development of efficient methods to synthesize non-canonical prolines and other chirally dense nitrogen heterocycles.

De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins.

Enzymes that bear a nonnative or artificially introduced metal center can engender novel reactivity and enable new spectroscopic and structural studies. In the case of metal-organic cofactors, such as metalloporphyrins, no general methods exist to build and incorporate new-to-nature cofactor analogs in vivo. We report here that a common laboratory strain, Escherichia coli BL21(DE3), biosynthesizes cobalt protoporphyrin IX (CoPPIX) under iron-limited, cobalt-rich growth conditions. In supplemented minimal media containing CoCl2, the metabolically produced CoPPIX is directly incorporated into multiple hemoproteins in place of native heme b (FePPIX). Five cobalt-substituted proteins were successfully expressed with this new-to-nature cobalt porphyrin cofactor: myoglobin H64V V68A, dye decolorizing peroxidase, aldoxime dehydratase, cytochrome P450 119, and catalase. We show conclusively that these proteins incorporate CoPPIX, with the CoPPIX making up at least 95% of the total porphyrin content. In cases in which the native metal ligand is a sulfur or nitrogen, spectroscopic parameters are consistent with retention of native metal ligands. This method is an improvement on previous approaches with respect to both yield and ease-of-implementation. Significantly, this method overcomes a long-standing challenge to incorporate nonnatural cofactors through de novo biosynthesis. By utilizing a ubiquitous laboratory strain, this process will facilitate spectroscopic studies and the development of enzymes for CoPPIX-mediated biocatalysis.

l-Threonine Transaldolase Activity Is Enabled by a Persistent Catalytic Intermediate.

l-Threonine transaldolases (lTTAs) are a poorly characterized class of pyridoxal-5'-phosphate (PLP) dependent enzymes responsible for the biosynthesis of diverse ß-hydroxy amino acids. Here, we study the catalytic mechanism of ObiH, an lTTA essential for biosynthesis of the ß-lactone natural product obafluorin. Heterologously expressed ObiH purifies as a mixture of chemical states including a catalytically inactive form of the PLP cofactor. Photoexcitation of ObiH promotes the conversion of the inactive state of the enzyme to the active form. UV-vis spectroscopic analysis reveals that ObiH catalyzes the retro-aldol cleavage of l-threonine to form a remarkably persistent glycyl quinonoid intermediate, with a half-life of ∼3 h. Protonation of this intermediate is kinetically disfavored, enabling on-cycle reactivity with aldehydes to form ß-hydroxy amino acids. We demonstrate the synthetic potential of ObiH via the single step synthesis of (2S,3R)-ß-hydroxyleucine. To further understand the structural features underpinning this desirable reactivity, we determined the crystal structure of ObiH bound to PLP as the Schiff's base at 1.66 Å resolution. This high-resolution model revealed a unique active site configuration wherein the evolutionarily conserved Asp that traditionally H-bonds to the cofactor is swapped for a neighboring Glu. Molecular dynamics simulations combined with mutagenesis studies indicate that a structural rearrangement is associated with l-threonine entry into the catalytic cycle. Together, these data explain the basis for the unique reactivity of lTTA enzymes and provide a foundation for future engineering and mechanistic analysis.

Modular control of l-tryptophan isotopic substitution via an efficient biosynthetic cascade.

Isotopologs are powerful tools for investigating biological systems. We report a biosynthetic-cascade synthesis of Trp isotopologs starting from indole, glycine, and formaldehyde using the enzymes l-threonine aldolase and an engineered ß-subunit of tryptophan synthase. This modular route to Trp isotopologs is simple and inexpensive, enabling facile access to these compounds.

Structure of a bound peptide phosphonate reveals the mechanism of nocardicin bifunctional thioesterase epimerase-hydrolase half-reactions.

Nonribosomal peptide synthetases (NRPSs) underlie the biosynthesis of many natural products that have important medicinal utility. Protection of the NRPS peptide products from proteolysis is critical to these pathways and is often achieved by structural modification, principally the introduction of D-amino acid residues into the elongating peptide. These amino acids are generally formed in situ from their L-stereoisomers by epimerization domains or dual-function condensation/epimerization domains. In singular contrast, the thioesterase domain of nocardicin biosynthesis mediates both the effectively complete L- to D-epimerization of its C-terminal amino acid residue (≥100:1) and hydrolytic product release. We report herein high-resolution crystal structures of the nocardicin thioesterase domain in ligand-free form and reacted with a structurally precise fluorophosphonate substrate mimic that identify the complete peptide binding pocket to accommodate both stereoisomers. These structures combined with additional functional studies provide detailed mechanistic insight into this unique dual-function NRPS domain.

Facile in Vitro Biocatalytic Production of Diverse Tryptamines.

Tryptamines are a medicinally important class of small molecules that serve as precursors to more complex, clinically used indole alkaloid natural products. Typically, tryptamine analogues are prepared from indoles through multistep synthetic routes. In the natural world, the desirable tryptamine synthon is produced in a single step by l-tryptophan decarboxylases (TDCs). However, no TDCs are known to combine high activity and substrate promiscuity, which might enable a practical biocatalytic route to tryptamine analogues. We have now identified the TDC from Ruminococcus gnavus as the first highly active and promiscuous member of this enzyme family. RgnTDC performs up to 96 000 turnovers and readily accommodates tryptophan analogues with substituents at the 4, 5, 6, and 7 positions, as well as alternative heterocycles, thus enabling the facile biocatalytic synthesis of >20 tryptamine analogues. We demonstrate the utility of this enzyme in a two-step biocatalytic sequence with an engineered tryptophan synthase to afford an efficient, cost-effective route to tryptamines from commercially available indole starting materials.

Engineered Biosynthesis of ß-Alkyl Tryptophan Analogues.

Noncanonical amino acids (ncAAs) with dual stereocenters at the α and ß positions are valuable precursors to natural products and therapeutics. Despite the potential applications of such bioactive ß-branched ncAAs, their availability is limited due to the inefficiency of the multistep methods used to prepare them. Herein we report a stereoselective biocatalytic synthesis of ß-branched tryptophan analogues using an engineered variant of Pyrococcus furiosus tryptophan synthase (PfTrpB), PfTrpB7E6 . PfTrpB7E6 is the first biocatalyst to synthesize bulky ß-branched tryptophan analogues in a single step, with demonstrated access to 27 ncAAs. The molecular basis for the efficient catalysis and broad substrate tolerance of PfTrpB7E6 was explored through X-ray crystallography and UV/Vis spectroscopy, which revealed that a combination of active-site and remote mutations increase the abundance and persistence of a key reactive intermediate. PfTrpB7E6 provides an operationally simple and environmentally benign platform for the preparation of ß-branched tryptophan building blocks.

Catalytic iron-carbene intermediate revealed in a cytochrome c carbene transferase.

Recently, heme proteins have been discovered and engineered by directed evolution to catalyze chemical transformations that are biochemically unprecedented. Many of these nonnatural enzyme-catalyzed reactions are assumed to proceed through a catalytic iron porphyrin carbene (IPC) intermediate, although this intermediate has never been observed in a protein. Using crystallographic, spectroscopic, and computational methods, we have captured and studied a catalytic IPC intermediate in the active site of an enzyme derived from thermostable Rhodothermus marinus (Rma) cytochrome c High-resolution crystal structures and computational methods reveal how directed evolution created an active site for carbene transfer in an electron transfer protein and how the laboratory-evolved enzyme achieves perfect carbene transfer stereoselectivity by holding the catalytic IPC in a single orientation. We also discovered that the IPC in Rma cytochrome c has a singlet ground electronic state and that the protein environment uses geometrical constraints and noncovalent interactions to influence different IPC electronic states. This information helps us to understand the impressive reactivity and selectivity of carbene transfer enzymes and offers insights that will guide and inspire future engineering efforts.