Impact of ancestral sequence reconstruction on mechanistic and structural enzymology

Ancestral sequence reconstruction (ASR) provides insight into the changes within a protein sequence across evolution. More specifically, it can illustrate how specific amino acid changes give rise to different phenotypes within a protein family. Over the last few decades it has established itself as a powerful technique for revealing molecular common denominators that govern enzyme function. Here, we describe the strength of ASR in unveiling catalytic mechanisms and emerging phenotypes for a range of different proteins, also highlighting biotechnological applications the methodology can provide.</p

Ancestral reconstruction of mammalian FMO1 enables structural determination, revealing unique features that explain its catalytic properties

Mammals rely on the oxidative flavin-containing monooxygenases (FMOs) to detoxify numerous and potentially deleterious xenobiotics; this activity extends to many drugs, giving FMOs high pharmacological relevance. However, our knowledge regarding these membrane-bound enzymes has been greatly impeded by the lack of structural information. We anticipated that ancestral-sequence reconstruction could help us identify protein sequences that are more amenable to structural analysis. As such, we hereby reconstructed the mammalian ancestral protein sequences of both FMO1 and FMO4, denoted as ancestral flavin-containing monooxygenase (AncFMO)1 and AncFMO4, respectively. AncFMO1, sharing 89.5% sequence identity with human FMO1, was successfully expressed as a functional enzyme. It displayed typical FMO activities as demonstrated by oxygenating benzydamine, tamoxifen, and thioanisole, drug-related compounds known to be also accepted by human FMO1, and both NADH and NADPH cofactors could act as electron donors, a feature only described for the FMO1 paralogs. AncFMO1 crystallized as a dimer and was structurally resolved at 3.0 Å resolution. The structure harbors typical FMO aspects with the flavin adenine dinucleotide and NAD(P)H binding domains and a C-terminal transmembrane helix. Intriguingly, AncFMO1 also contains some unique features, including a significantly porous and exposed active site, and NADPH adopting a new conformation with the 2'-phosphate being pushed inside the NADP+ binding domain instead of being stretched out in the solvent. Overall, the ancestrally reconstructed mammalian AncFMO1 serves as the first structural model to corroborate and rationalize the catalytic properties of FMO1

In vitro construction of the COQ metabolon unveils the molecular determinants of coenzyme Q biosynthesis

Metabolons are protein assemblies that perform a series of reactions in a metabolic pathway. However, the general importance and aptitude of metabolons for enzyme catalysis remain poorly understood. In animals, biosynthesis of coenzyme Q is currently attributed to ten different proteins, with COQ3, COQ4, COQ5, COQ6, COQ7 and COQ9 forming the iconic COQ metabolon. Yet several reaction steps conducted by the metabolon remain enigmatic. To elucidate the prerequisites for animal coenzyme Q biosynthesis, we sought to construct the entire metabolon in vitro. Here we show that this approach, rooted in ancestral sequence reconstruction, reveals the enzymes responsible for the uncharacterized steps and captures the biosynthetic pathway in vitro. We demonstrate that COQ8, a kinase, increases and streamlines coenzyme Q production. Our findings provide crucial insight into how biocatalytic efficiency is regulated and enhanced by these biosynthetic engines in the context of the cell. (Figure presented.).</p

Evolutionary and structural analyses of the NADPH oxidase family in eukaryotes reveal an initial calcium dependency

Reactive oxygen species are unstable molecules generated by the partial reduction of dioxygen. NADPH oxidases are a ubiquitous family of enzymes devoted to ROS production. They fuel an array of physiological roles in different species and are chemically demanding enzymes requiring FAD, NADPH and heme prosthetic groups in addition to either calcium or a various number of cytosolic mediators for activity. These activating partners are exclusive components that partition and distinguish the NOX members from one another. To gain insight into the evolution of these activating mechanisms, and in general in their evolutionary history, we conducted an in-depth phylogenetic analysis of the NADPH oxidase family in eukaryotes. We show that all characterized NOXs share a common ancestor, which comprised a fully formed catalytic unit. Regarding the activation mode, we identified calcium-dependency as the earliest form of NOX regulation. The protein-protein mode of regulation would have evolved more recently by gene-duplication with the concomitant loss of the EF-hands motif region. These more recent events generated the diversely activated NOX systems as observed in extant animals and fungi

Ancestral-sequence reconstruction unveils the structural basis of function in mammalian FMOs

Flavin-containing monooxygenases (FMOs) are ubiquitous in all domains of life and metabolize a myriad of xenobiotics, including toxins, pesticides and drugs. However, despite their pharmacological importance, structural information remains bereft. To further our understanding behind their biochemistry and diversity, we used ancestral-sequence reconstruction, kinetic and crystallographic techniques to scrutinize three ancient mammalian FMOs: AncFMO2, AncFMO3-6 and AncFMO5. Remarkably, all AncFMOs could be crystallized and were structurally resolved between 2.7- and 3.2-Å resolution. These crystal structures depict the unprecedented topology of mammalian FMOs. Each employs extensive membrane-binding features and intricate substrate-profiling tunnel networks through a conspicuous membrane-adhering insertion. Furthermore, a glutamate–histidine switch is speculated to induce the distinctive Baeyer–Villiger oxidation activity of FMO5. The AncFMOs exhibited catalysis akin to human FMOs and, with sequence identities between 82% and 92%, represent excellent models. Our study demonstrates the power of ancestral-sequence reconstruction as a strategy for the crystallization of proteins.Fil: Nicoll, Callum R.. Universita Degli Studi Di Pavia; ItaliaFil: Bailleul, Gautier. University of Groningen; Países BajosFil: Fiorentini, Filippo. Universita Degli Studi Di Pavia; ItaliaFil: Mascotti, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Fraaije, Marco Wilhelmus. University Of Groningen. Faculty Of Science And Engineering. Engineering And Technology Institute Groningen.; Países BajosFil: Mattevi, Andrea. Universita Degli Studi Di Pavia; Itali

Evolution of enzyme functionality in the flavin-containing monooxygenases

Among the molecular mechanisms of adaptation in biology, enzyme functional diversification is indispensable. By allowing organisms to expand their catalytic repertoires and adopt fundamentally different chemistries, animals can harness or eliminate new-found substances and xenobiotics that they are exposed to in new environments. Here, we explore the flavin-containing monooxygenases (FMOs) that are essential for xenobiotic detoxification. Employing a paleobiochemistry approach in combination with enzymology techniques we disclose the set of historical substitutions responsible for the family's functional diversification in tetrapods. Remarkably, a few amino acid replacements differentiate an ancestral multi-tasking FMO into a more specialized monooxygenase by modulating the oxygenating flavin intermediate. Our findings substantiate an ongoing premise that enzymatic function hinges on a subset of residues that is not limited to the active site core

Publisher Correction:Ancestral-sequence reconstruction unveils the structural basis of function in mammalian FMOs (Nature Structural &amp; Molecular Biology, (2020), 27, 1, (14-24), 10.1038/s41594-019-0347-2)

In the print and PDF versions of this article initially published, panel e was missing from Fig. 6. The error has been corrected in the PDF version of the article.(Figure presented.)