@article{HeNoorRamosParraetal.2020, author = {He, Hai and Noor, Elad and Ramos-Parra, Perla A. and Garc{\´i}a-Valencia, Liliana E. and Patterson, Jenelle A. and D{\´i}az de la Garza, Roc{\´i}o I. and Hanson, Andrew D. and Bar-Even, Arren}, title = {In Vivo Rate of Formaldehyde Condensation with Tetrahydrofolate}, series = {Metabolites}, volume = {10}, journal = {Metabolites}, number = {65}, publisher = {MDPI}, address = {Basel}, issn = {2218-1989}, doi = {10.3390/metabo10020065}, pages = {15}, year = {2020}, 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.}, language = {en} } @article{PattersonHeFolzetal.2020, author = {Patterson, Jenelle A. and He, Hai and Folz, Jacob S. and Li, Qiang and Wilson, Mark A. and Fiehn, Oliver and Bruner, Steven D. and Bar-Even, Arren and Hanson, Andrew D.}, title = {Thioproline formation as a driver of formaldehyde toxicity in Escherichia coli}, series = {Biochemical Journal}, volume = {477}, journal = {Biochemical Journal}, number = {9}, publisher = {Portland Press}, address = {London}, issn = {1470-8728}, doi = {10.1042/BCJ20200198}, pages = {1745 -- 1757}, year = {2020}, abstract = {Formaldehyde (HCHO) is a reactive carbonyl compound that formylates and cross-links proteins, DNA, and small molecules. It is of specific concern as a toxic intermediate in the design of engineered pathways involving methanol oxidation or formate reduction. The interest in engineering these pathways is not, however, matched by engineering-relevant information on precisely why HCHO is toxic or on what damage-control mechanisms cells deploy to manage HCHO toxicity. The only well-defined mechanism for managing HCHO toxicity is formaldehyde dehydrogenase-mediated oxidation to formate, which is counterproductive if HCHO is a desired pathway intermediate. We therefore sought alternative HCHO damage-control mechanisms via comparative genomic analysis. This analysis associated homologs of the Escherichia coli pepP gene with HCHO-related one-carbon metabolism. Furthermore, deleting pepP increased the sensitivity of E. coli to supplied HCHO but not other carbonyl compounds. PepP is a proline aminopeptidase that cleaves peptides of the general formula X-Pro-Y, yielding X + Pro-Y. HCHO is known to react spontaneously with cysteine to form the close proline analog thioproline (thiazolidine-4-carboxylate), which is incorporated into proteins and hence into proteolytic peptides. We therefore hypothesized that certain thioproline-containing peptides are toxic and that PepP cleaves these aberrant peptides. Supporting this hypothesis, PepP cleaved the model peptide Ala-thioproline-Ala as efficiently as Ala-Pro-Ala in vitro and in vivo, and deleting pepP increased sensitivity to supplied thioproline. Our data thus (i) provide biochemical genetic evidence that thioproline formation contributes substantially to HCHO toxicity and (ii) make PepP a candidate damage-control enzyme for engineered pathways having HCHO as an intermediate.}, language = {en} } @misc{HeNoorRamosParraetal.2020, author = {He, Hai and Noor, Elad and Ramos-Parra, Perla A. and Garc{\´i}a-Valencia, Liliana E. and Patterson, Jenelle A. and D{\´i}az de la Garza, Roc{\´i}o I. and Hanson, Andrew D. and Bar-Even, Arren}, title = {In Vivo Rate of Formaldehyde Condensation with Tetrahydrofolate}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {998}, issn = {1866-8372}, doi = {10.25932/publishup-47647}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-476472}, pages = {17}, year = {2020}, 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.}, language = {en} }