2019 FMO breakthrough : Scientists contruct FMO structure

Probably all living things have at least 1 FMO (Flavin mono-oxygenase enzyme).

Humans have 5 (and a number of pseudogenes), with FMO3 deemed the main 'worker' in humans.

Although FMOs were hypothesized in the 60s, and proven around 1980, it has been impossible to construct FMO in a lab until 2019.

A group led by a Dutch lab managed to do so and publish a paper in Dec 2019.

They have also updated the paper.

While it is a breakthrough, it's not currently known how this will help with 'FMO3 Malodor' 

1. Will it mean more interest and research ? 

2. Does it help in a practical way ?

While it might help pharma companies to make drugs (that are worse for sub-FMO3 people), it's not clear if any benefit will come to sub-FMO3 people.

Keeping in mind that probably the majority of people probably carry at least 1 FMO3 variant (currently taught as benign).   

Dec 2019 paper :

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

 (Link to abstract)

Lead Researcher : Marco W. Fraaije 

Fraaije Lab, Groningen Uni

Quotes from paper

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.

 

Press Release quotes :

To disarm toxic substances, many organisms – including humans – possess enzymes called flavin-containing monooxygenases (FMOs). Despite their importance, the structure of the enzymes has not been resolved, as the protein is too unstable to study in detail. University of Groningen enzyme engineer Marco Fraaije and colleagues from Italy and Argentina reconstructed the ancestral genetic sequences for three FMO genes and subsequently studied the enzymes. The ancestral enzymes proved to be stable enough to study and revealed how FMOs metabolize toxic substances. The results were published on 23 December in Nature Structural & Molecular Biology...

FMOs are present in animals, plants and bacteria. In humans, our five different FMOs are not only involved in metabolizing toxic substances but also in drug activation, while mutations in FMO genes may cause illnesses. ‘These enzymes are studied in detail by pharmaceutical companies but we still had no detailed structure available,’ says Fraaije. Human FMOs are membrane-bound proteins, which proved to be impossible to crystallize for standard structural analysis by X-ray diffraction. ‘My group published the structure of a bacterial FMO, some fifteen years ago, but this was not a membrane-bound protein.’...

...‘The results are fascinating,’ says Fraaije. ‘The membrane-bound part of the enzyme forms a kind of tunnel through which substances can be transported to the active site.’ Many toxic compounds are fatty substances that will accumulate in the fatty cell membrane. ‘The FMO enzymes can take them from the membrane and oxidize them.’ This makes the toxins more hydrophilic, which makes it easier for the cell to excrete them. Whereas the active site is the same in the three FMOs, they have slightly different tunnels, probably suited to different classes of toxic compounds. ‘We knew that different FMOs metabolize different substances and now we can explain why this is so.’...

...Scientists and pharmaceutical companies are now finally able to see how the FMOs work. ‘This could help in the design of drugs that are activated by these enzymes. And the observation that the ancestral protein is more stable is also of interest...

... Finally, it is now possible to reconstruct the effect of disease-causing mutations in FMO genes. One of those mutations causes fish odour syndrome, where a mutation in FMO3 results in the inability to metabolize the substance trimethylamine. This substance, which has a strong fish odour, consequently builds up in the body and is released in sweat, urine and breath, among other things . Fraaije: ‘This was a high-risk project, as we didn’t know if the ancestral protein would be stable enough. But it has paid off.’

press release links :

Groningen Uni

Eurekalert

Prof Elizabeth Shephard and Prof Ian Phillips comment on breakthrough (quotes)

link

FMOs catalyze the oxidative metabolism of a range of chemicals, including drugs, pesticides and compounds derived from the diet by the action of gut bacteria1,2,3,4. The enzymes act at the interface between an organism and its chemical environment to protect against potentially harmful foreign chemicals. Some FMOs are also involved in endogenous metabolic processes: in the regulation of energy balance5, metabolic aging6 and glucose homeostasis7. In vertebrates, FMOs are located in the membranes of the endoplasmic reticulum and have proved, to date, impossible to crystallize. Now, based on the reconstruction of ancestral mammalian FMO sequences, Nicoll et al.8 report the crystallization and structures of three membrane-bound FMOs. This major advance provides a structural basis for the catalytic mechanism of FMOs and gives insights into how the enzymes bind to membranes and control access to their catalytic site.

Early during the evolution of tetrapods, duplications of a single FMO gene gave rise to a family of genes. In humans, the FMO gene family consists of five functional genes (FMO1, 2, 3, 4 and 5) and six pseudogenes9, one of which is FMO6. FMO3 and FMO6 arose from a more recent duplication that took place early during mammalian evolution9. Based on a phylogenetic analysis, Nicoll et al. reconstructed ancestral sequences for three mammalian FMOs: FMO2, FMO5 and the precursor of FMO3 and 6. The ancestral FMOs, which have 83–92% amino acid sequence identity with the corresponding extant human FMO, were expressed in Escherichia coli as holoenzymes bound to their prosthetic group FAD. The ancestral FMOs were catalytically active, with kinetic parameters similar to those of the corresponding human FMO, and ancestral FMOs 2 and 3/6 reacted rapidly with oxygen to form a stable C4a hydroperoxyflavin intermediate, a key feature of the catalytic mechanism

Remarkably, despite numerous unsuccessful attempts to crystallize human FMO3 and FMO5, all three of the ancestral proteins crystallized, possibly because of their higher melting temperatures. The crystal structures of the ancestral FMOs provide valuable insights into how FMOs bind to membranes, interact with FAD and the cofactor NADP+, and control access to their catalytic sites...

...The availability of structures of ancestral mammalian FMOs will enable more accurate modeling of structures of extant human FMOs. For instance, a structure of human FMO3 would contribute to understanding the effect on enzyme activity of known causative mutations of trimethylaminuria and help predict the likely consequences of novel mutations. Such structures would also provide a basis for structure-based design of drugs that are substrates of FMOs. 

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