Abstract
Formaldehyde is a highly reactive compound that participates in multiple spontaneous reactions, but these are mostly deleterious and damage cellular components. In contrast, the spontaneous condensation of formaldehyde with tetrahydrofolate (THF) has been proposed to contribute to the assimilation of this intermediate during growth on C1 carbon sources such as methanol. However, the in vivo rate of this condensation reaction is unknown and its possible contribution to growth remains elusive. Here, we used microbial platforms to assess the rate of this condensation in the cellular environment. We constructed Escherichia coli strains lacking the enzymes that naturally produce 5,10-methylene-THF. These strains were able to grow on minimal medium only when equipped with a sarcosine (N-methyl-glycine) oxidation pathway that sustained a high cellular concentration of formaldehyde, which spontaneously reacts with THF to produce 5,10-methylene-THF. We used flux balance analysis to derive the rate of the spontaneous condensation from the observed growth rate. According to this, we calculated that a microorganism obtaining its entire biomass via the spontaneous condensation of formaldehyde with THF would have a doubling time of more than three weeks. Hence, this spontaneous reaction is unlikely to serve as an effective route for formaldehyde assimilation.
Highlights
Most metabolic conversions are catalyzed by dedicated enzymes or by promiscuous enzymes that evolved to catalyze other reactions [1]
We construct microbial platforms to directly assess the in vivo condensation rate of formaldehyde and THF
Reviewing E. coli native enzymes, we identified two that might be able to catalyze the condensation reaction promiscuously: (i) 3-methyl-2-oxobutanoate hydroxymethyltransferase (PanB), some variants of which are known to condense formaldehyde and 3-methyl-2-oxobutanoate to give 2-dehydropantoate [26], which could donate a C1 moiety to THF via the reversal of the major enzyme activity. (ii) Threonine aldolase (LtaE), an enzyme evolutionarily related to serine hydroxymethyltransferase, which might be able to promiscuously accept formaldehyde and THF [27]
Summary
Most metabolic conversions are catalyzed by dedicated enzymes or by promiscuous enzymes that evolved to catalyze other reactions [1]. Condensation of formaldehyde with tetrahydromethanopterin, which plays a key role in the methylotrophic serine cycle [7], was once thought to proceed only spontaneously, but later an enzyme catalyzing this reaction was identified (‘formaldehyde-activating enzyme’, Fae) [8]. Another spontaneously occurring formaldehyde condensation reaction occurs with the universal. The kinetics of the spontaneous condensation of formaldehyde and THF have been measured in vitro [13], the in vivo reaction rate—that is, reaction kinetics under physiological conditions and concentrations—have never been measured It remains unclear whether this spontaneous reaction can play a physiological role in formaldehyde metabolism and assimilation.
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