Engineering enzymes with non-canonical active site functionality

The combination of computational enzyme design and laboratory evolution provides an attractive platform for the creation of protein catalysts with new function. To date, designed mechanisms have relied upon Nature’s alphabet of 20 genetically encoded amino acids, which greatly restricts the range of functionality which can be installed into enzyme active sites. Here, we have exploited engineered components of the cellular translation machinery to create a protein catalyst which operates via a non-canonical catalytic nucleophile. We have subsequently shown that powerful laboratory evolution protocols can be readily adapted to allow optimization of enzymes containing non-canonical active site functionality. Crystal structures obtained along the evolutionary trajectory highlight the origins of improved activity. Thus our approach merges beneficial features of organo- and biocatalysis, by combining the intrinsic reactivities and greater versatility of small molecule catalysts with the rate enhancements, reaction selectivities and evolvability of proteins. Please click Additional Files below to see the full abstract

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

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

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

[Image: see text] 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

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

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

International audienceThe 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