Elucidation of the stereochemical mechanism of cystathionine γ-lyase reveals how substrate specificity constrains catalysis.

Pyridoxal phosphate (PLP)-dependent enzymes play essential roles in metabolism and have found applications for organic synthesis and as enzyme therapeutics. The vinylglycine ketimine (VGK) subfamily hosts a growing set of enzymes that play diverse roles in primary and secondary metabolism. However, the molecular determinates of substrate specificity and the complex acid-base chemistry that enables VGK catalysis remain enigmatic.

Biocatalytic asymmetric aldol addition into unactivated ketones.

Enzymes are renowned for their catalytic efficiency and selectivity, but many classical transformations in organic synthesis have no biocatalytic counterpart. Aldolases are prodigious C-C bond-forming enzymes, but their reactivity has only been extended past activated carbonyl electrophiles in special cases. To probe the mechanistic origins of this limitation, we use a pair of aldolases whose activity is dependent on pyridoxal phosphate.

Substrate-Multiplexed Assessment of Aromatic Prenyltransferase Activity.

An increasingly effective strategy to identify synthetically useful enzymes is to sample the diversity already present in Nature. Here, we construct and assay a panel of phylogenetically diverse aromatic prenyltransferases (PTs). These enzymes catalyze a variety of C-C bond forming reactions in natural product biosynthesis and are emerging as tools for synthetic chemistry and biology.

Molecular Determinants of Efficient Cobalt-Substituted Hemoprotein Production in .

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 BL21(DE3). This discovery inspired us to explore the determinants of metal specificity for metallocofactor biosynthesis in .

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.

Engineered Biocatalytic Synthesis of β-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 β-substituted amino acids. However, there are few examples of biocatalytic N-substitution reactions to make α,β-diamino acids.

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.

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.

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.

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.

Investigation of β-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 β-substitution capability of the OASS from Citrullus vulgaris (CvOASS), a putative l-mimosine synthase.

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.

Scalable and Selective β-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 β-hydroxy-α-amino acids, which are important synthetic building blocks.

Asymmetric Alkylation of Ketones Catalyzed by Engineered TrpB.

The β-subunit of tryptophan synthase (TrpB) catalyzes a PLP-mediated β-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.

De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in 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, BL21(DE3), biosynthesizes cobalt protoporphyrin IX (CoPPIX) under iron-limited, cobalt-rich growth conditions.

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 β-hydroxy amino acids. Here, we study the catalytic mechanism of ObiH, an lTTA essential for biosynthesis of the β-lactone natural product obafluorin. Heterologously expressed ObiH purifies as a mixture of chemical states including a catalytically inactive form of the PLP cofactor.

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 β-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.

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).

Engineered Biosynthesis of β-Alkyl Tryptophan Analogues.

Noncanonical amino acids (ncAAs) with dual stereocenters at the α and β positions are valuable precursors to natural products and therapeutics. Despite the potential applications of such bioactive β-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 β-branched tryptophan analogues using an engineered variant of Pyrococcus furiosus tryptophan synthase (PfTrpB), PfTrpB .

Catalytic iron-carbene intermediate revealed in a cytochrome 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 () cytochrome 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.

Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble.

Allosteric enzymes contain a wealth of catalytic diversity that remains distinctly underutilized for biocatalysis. Tryptophan synthase is a model allosteric system and a valuable enzyme for the synthesis of noncanonical amino acids (ncAA). Previously, we evolved the β-subunit from Pyrococcus furiosus, PfTrpB, for ncAA synthase activity in the absence of its native partner protein PfTrpA.