N- and S-oxygenation activity of truncated human flavin-containing monooxygenase 3 and its common polymorphic variants

Abstract

Human flavin-containing monooxygenase 3 (FMO3) is a membrane-bound, phase I drug metabolizing enzyme. It is highly polymorphic with some of its variants demonstrating differences in rates of turnover of its substrates: xenobiotics including drugs as well as dietary compounds. In order to measure its in vitro activity and compare any differences between the wild type enzyme and its polymorphic variants, we undertook a systematic study using different engineered proteins, heterologously expressed in bacteria, purified and catalytically characterized with 3 different substrates. These included the full-length as well as the more soluble C-terminal truncated versions of the common polymorphic variants (E158K, V257M and E308G) of FMO3 in addition to the fulllength and truncated wild-type proteins. In vitro activity assays were performed with benzydamine, tamoxifen and sulindac sulfide, whose products were measured by HPLC. Differences in catalytic properties between the wild-type FMO3 and its common polymorphic variants were similar to those observed with the truncated, more soluble versions of the enzymes. Interestingly, the truncated enzymes were better catalysts than the full-length proteins. The data obtained point to the feasibility of using the more soluble forms of this enzyme for in vitro drug assays as well as future biotechnological applications possibly in high throughput systems such as bioelectrochemical platforms and biosensors.

Adaptive Modelling of Mutated FMO3 Enzyme Could Unveil Unexplored Scenarios Linking Variant Haplotypes to TMAU Phenotypes

“…The situation resulted completely different for FMO3 carrying Y331stop and P380Fs variants in haplotypes (P380Fs + E308G and P380Fs + P153L + E158K, respectively). It is already known from the literature that both variants lead to a truncated protein whose enzymatic activity is reduced or totally absent, depending on whether this variant is in a heterozygous or a homozygous condition [27]. However, the molecular mechanisms underlying this deficit are not yet fully known.…”

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

“…Tamoxifen and its metabolite were separated by isocratic elution and were detected at a wavelength of 276 nm. Mobile phase consisted of 82% of methanol and 18% of 1% triethylamine, analysis time was 20 min with a flow rate of 1.1 ml/min [33]. Retention time of tamoxifen and its N-oxide product were 6.5 min and 3.5 min, respectively.…”

A multi-channel microfluidic platform based on human flavin-containing monooxygenase 3 for personalised medicine

“…21 In particular, FMO3 is the most important isoform present in the adult liver, and it is known to exhibit single nucleotide polymorphisms (SNPs). [22][23][24] SNPs can have a major impact on the pharmacokinetics and pharmacodynamics of a particular drug, and therefore contribute to variations in susceptibility and toxicity in individuals carrying them. For this reason, a better and more personalised treatment with tailored dosages could avoid not only treatment failure but also adverse reactions leading to toxicity.…”

“…Tamoxifen, an antiestrogen medication widely used in breast cancer therapy and chemoprevention, was chosen as a model drug, since it is a known substrate of FMO3. 23,24,29,30 The chip was designed with a single inlet (connected to a syringe pump) and four separate channels so that the solution containing the drug is equally split to reach each immobilised enzyme at the same time. The channels were drawn with a serpentine shape to increase the available surface area for enzyme immobilization.…”

“…Tamoxifen, an antiestrogen medication widely used in breast cancer therapy and chemoprevention, was chosen as a model drug, since it is a known substrate of FMO3. 23,24,29,30…”