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

Abstract

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.

Human flavin-containing monooxygenase 1 and its long-sought hydroperoxyflavin intermediate

“…The structural integrity and folding of the pure protein were estimated by the circular dichroism (Figure 1). The fitting of the obtained spectra by the β-structure selection (BeStSel) algorithm [31] allowed to predict hFMO1 secondary structure content leading to a 37% alpha helical content in agreement with previously published data for the human FMO family [27,28].…”

“…Reduced hFMO1 was rapidly mixed with oxygenated buffer, and the formation and decay of the C4a-hydroperoxyflavin intermediate was followed at 368 nm [23,28]. Moreover, the effect of the presence of the substrate on the re-oxidation process was investigated in the absence and presence of the substrate hypotaurine.…”

“…Association to membrane is a well-known hinderance to the general crystallization process of proteins. To overcome this obstacle, synthetic genes coding for ancestral FMO isoforms and in particular the FMO1 was recently synthesized and the resulting synthetic protein was crystalized [28]. In this work the second option was followed in order to investigate the reactivity of this intermediate in hFMO1 and compare its behaviour to other human FMOs as well as its homologous counterpart, pFMO1.…”

Efficient Exploration of Sequence Space by Sequence-Guided Protein Engineering and Design

“…ASR has now been successfully applied to a variety of protein families to engineer remarkably thermostable biocatalysts, − biopharmaceuticals, − and research tools, − including carboxylic acid reductases ( T m ≤35 °C higher than those of characterized extant proteins), amino acid binding proteins (30 °C), ketol-acid reductoisomerases (30 °C), haloalkane dehalogenases (24 °C), , and diterpene cyclases (13 °C) . Surprisingly, major improvements in thermostability have been observed even when reconstructing more evolutionarily recent proteins that are not predicted to have originated from thermophilic organisms; ,, for example, reconstructed cytochrome P450 enzymes and flavin-containing monooxygenases putatively derived from ancestral vertebrates showed T m values ≤30 and ≤22 °C higher than those of extant homologues, respectively. , Although the origin of stabilizing mutations in such cases is not entirely understood, systematic biases in the commonly used maximum likelihood method for ASR may be partly responsible; − for example, sequence similarity between ancestral and consensus sequences based on the same sequence data set has suggested a bias of ASR toward the consensus sequence at ambiguously reconstructed positions, , although this bias cannot fully explain the higher stability of ancestral proteins compared with that of extant proteins. , There is a need to better understand the source of stabilizing mutations in reconstructed ancestral sequences to predict which protein families will be amenable to ASR as a method for engineering thermostability and to guide the choice of ancestral nodes for experimental characterization; nonetheless, the examples listed above provide empirical evidence that ASR can be used to substantially increase protein thermostability when applied to a data set of sufficient sequence diversity, even if the resulting ancestral sequences are not evolutionarily ancient (<300 million years old).…”

Ancestral Sequence Reconstruction Enhances Gene Mining Efforts for Industrial Ene Reductases by Expanding Enzyme Panels with Thermostable Catalysts

“…Ancestral sequence reconstruction (ASR) is a method that allows the inference of “ancestral” genes from a collection of homologous wild-type genes. − Previously, ASR has been applied to diverse enzyme families such as L-amino acid oxidases, , carboxylic acid reductases, 3-isopropylmalate dehydrogenases, transaminases, serum paraoxonases, β-lactamases, , cytochromes P450, terpene cyclases, − and mammalian flavin-containing monooxygenases . These studies have demonstrated that ASR can be used to produce novel, active enzyme sequences and that ASR-derived enzymes are often more thermostable than their wild-type (WT) counterparts. ,, The improved stability of ASR enzymes has led to interest in ASR as a tool for developing industrial biocatalysts, but it is not clear if ASR enzymes constitute a superior source of industrial enzymes compared to WT enzymes.…”