Design of Heme Enzymes with a Tunable Substrate Binding Pocket Adjacent to an Open Metal Coordination Site.

The catalytic versatility of pentacoordinated iron is highlighted by the broad range of natural and engineered activities of heme enzymes such as cytochrome P450s, which position a porphyrin cofactor coordinating a central iron atom below an open substrate binding pocket. This catalytic prowess has inspired efforts to design de novo helical bundle scaffolds that bind porphyrin cofactors. However, such designs lack the large open substrate binding pocket of P450s, and hence, the range of chemical transformations accessible is limited. Here, with the goal of combining the advantages of the P450 catalytic site geometry with the almost unlimited customizability of de novo protein design, we design a high-affinity heme-binding protein, dnHEM1, with an axial histidine ligand, a vacant coordination site for generating reactive intermediates, and a tunable distal pocket for substrate binding. A 1.6 Å X-ray crystal structure of dnHEM1 reveals excellent agreement to the design model with key features programmed as intended. The incorporation of distal pocket substitutions converted dnHEM1 into a proficient peroxidase with a stable neutral ferryl intermediate. In parallel, dnHEM1 was redesigned to generate enantiocomplementary carbene transferases for styrene cyclopropanation (up to 93% isolated yield, 5000 turnovers, 97:3 e.r.) by reconfiguring the distal pocket to accommodate calculated transition state models. Our approach now enables the custom design of enzymes containing cofactors adjacent to binding pockets with an almost unlimited variety of shapes and functionalities.

Engineering enzyme activity using an expanded amino acid alphabet.

Enzyme design and engineering strategies are typically constrained by the limited size of nature's genetic alphabet, comprised of only 20 canonical amino acids. In recent years, site-selective incorporation of non-canonical amino acids (ncAAs) via an expanded genetic code has emerged as a powerful means of inserting new functional components into proteins, with hundreds of structurally diverse ncAAs now available. Here, we highlight how the emergence of an expanded repertoire of amino acids has opened new avenues in enzyme design and engineering. ncAAs have been used to probe complex biological mechanisms, augment enzyme function and, most ambitiously, embed new catalytic mechanisms into protein active sites that would be challenging to access within the constraints of nature's genetic code. We predict that the studies reviewed in this article, along with further advances in genetic code expansion technology, will establish ncAA incorporation as an increasingly important tool for biocatalysis in the coming years.

A Noncanonical Tryptophan Analogue Reveals an Active Site Hydrogen Bond Controlling Ferryl Reactivity in a Heme Peroxidase.

Nature employs high-energy metal-oxo intermediates embedded within enzyme active sites to perform challenging oxidative transformations with remarkable selectivity. Understanding how different local metal-oxo coordination environments control intermediate reactivity and catalytic function is a long-standing objective. However, conducting structure-activity relationships directly in active sites has proven challenging due to the limited range of amino acid substitutions achievable within the constraints of the genetic code. Here, we use an expanded genetic code to examine the impact of hydrogen bonding interactions on ferryl heme structure and reactivity, by replacing the N-H group of the active site Trp51 of cytochrome c peroxidase by an S atom. Removal of a single hydrogen bond stabilizes the porphyrin π-cation radical state of CcP W191F compound I. In contrast, this modification leads to more basic and reactive neutral ferryl heme states, as found in CcP W191F compound II and the wild-type ferryl heme-Trp191 radical pair of compound I. This increased reactivity manifests in a >60-fold activity increase toward phenolic substrates but remarkably has negligible effects on oxidation of the biological redox partner cytc. Our data highlight how Trp51 tunes the lifetimes of key ferryl intermediates and works in synergy with the redox active Trp191 and a well-defined substrate binding site to regulate catalytic function. More broadly, this work shows how noncanonical substitutions can advance our understanding of active site features governing metal-oxo structure and reactivity.

Rewiring the "Push-Pull" Catalytic Machinery of a Heme Enzyme Using an Expanded Genetic Code.

Nature employs a limited number of genetically encoded axial ligands to control diverse heme enzyme activities. Deciphering the functional significance of these ligands requires a quantitative understanding of how their electron-donating capabilities modulate the structures and reactivities of the iconic ferryl intermediates compounds I and II. However, probing these relationships experimentally has proven to be challenging as ligand substitutions accessible via conventional mutagenesis do not allow fine tuning of electron donation and typically abolish catalytic function. Here, we exploit engineered translation components to replace the histidine ligand of cytochrome c peroxidase (CcP) by a less electron-donating N δ-methyl histidine (Me-His) with little effect on the enzyme structure. The rate of formation (k 1) and the reactivity (k 2) of compound I are unaffected by ligand substitution. In contrast, proton-coupled electron transfer to compound II (k 3) is 10-fold slower in CcP Me-His, providing a direct link between electron donation and compound II reactivity, which can be explained by weaker electron donation from the Me-His ligand ("the push") affording an electron-deficient ferryl oxygen with reduced proton affinity ("the pull"). The deleterious effects of the Me-His ligand can be fully compensated by introducing a W51F mutation designed to increase "the pull" by removing a hydrogen bond to the ferryl oxygen. Analogous substitutions in ascorbate peroxidase lead to similar activity trends to those observed in CcP, suggesting that a common mechanistic strategy is employed by enzymes using distinct electron transfer pathways. Our study highlights how noncanonical active site substitutions can be used to directly probe and deconstruct highly evolved bioinorganic mechanisms.

Design and evolution of an enzyme with a non-canonical organocatalytic mechanism.

The combination of computational design and laboratory evolution is a powerful and potentially versatile strategy for the development of enzymes with new functions1-4. However, the limited functionality presented by the genetic code restricts the range of catalytic mechanisms that are accessible in designed active sites. Inspired by mechanistic strategies from small-molecule organocatalysis5, here we report the generation of a hydrolytic enzyme that uses Nδ-methylhistidine as a non-canonical catalytic nucleophile. Histidine methylation is essential for catalytic function because it prevents the formation of unreactive acyl-enzyme intermediates, which has been a long-standing challenge when using canonical nucleophiles in enzyme design6-10. Enzyme performance was optimized using directed evolution protocols adapted to an expanded genetic code, affording a biocatalyst capable of accelerating ester hydrolysis with greater than 9,000-fold increased efficiency over free Nδ-methylhistidine in solution. Crystallographic snapshots along the evolutionary trajectory highlight the catalytic devices that are responsible for this increase in efficiency. Nδ-methylhistidine can be considered to be a genetically encodable surrogate of the widely employed nucleophilic catalyst dimethylaminopyridine11, and its use will create opportunities to design and engineer enzymes for a wealth of valuable chemical transformations.

An oxidative N-demethylase reveals PAS transition from ubiquitous sensor to enzyme.

The universal Per-ARNT-Sim (PAS) domain functions as a signal transduction module involved in sensing diverse stimuli such as small molecules, light, redox state and gases. The highly evolvable PAS scaffold can bind a broad range of ligands, including haem, flavins and metal ions. However, although these ligands can support catalytic activity, to our knowledge no enzymatic PAS domain has been found. Here we report characterization of the first PAS enzyme: a haem-dependent oxidative N-demethylase. Unrelated to other amine oxidases, this enzyme contains haem, flavin mononucleotide, 2Fe-2S and tetrahydrofolic acid cofactors, and specifically catalyses the NADPH-dependent oxidation of dimethylamine. The structure of the α subunit reveals that it is a haem-binding PAS domain, similar in structure to PAS gas sensors. The dimethylamine substrate forms part of a highly polarized oxygen-binding site, and directly assists oxygen activation by acting as both an electron and proton donor. Our data reveal that the ubiquitous PAS domain can make the transition from sensor to enzyme, suggesting that the PAS scaffold can support the development of artificial enzymes.

Identification of a Class of Protein ADP-Ribosylating Sirtuins in Microbial Pathogens.

Sirtuins are an ancient family of NAD(+)-dependent deacylases connected with the regulation of fundamental cellular processes including metabolic homeostasis and genome integrity. We show the existence of a hitherto unrecognized class of sirtuins, found predominantly in microbial pathogens. In contrast to earlier described classes, these sirtuins exhibit robust protein ADP-ribosylation activity. In our model organisms, Staphylococcus aureus and Streptococcus pyogenes, the activity is dependent on prior lipoylation of the target protein and can be reversed by a sirtuin-associated macrodomain protein. Together, our data describe a sirtuin-dependent reversible protein ADP-ribosylation system and establish a crosstalk between lipoylation and mono-ADP-ribosylation. We propose that these posttranslational modifications modulate microbial virulence by regulating the response to host-derived reactive oxygen species.