Acetyl-CoA Production and Utilization during Growth of the Facultative Methylotroph Pseudomonas AM1 on Ethanol, Malonate and 3-Hydroxybutyrate

Abstract

In Pseudomonas AM I , conversion of 3-hydroxybutyrate to acetyl-CoA is mediated by an inducible 3-hydroxybutyrate dehydrogenase, an acetoacetate : succinate coenzyme A transferase (specific for succinyl-CoA) and an inducible /I-ketothiolase. Ethanol is oxidized to acetate by the same enzymes as are involved in methanol oxidation to formate. An inducible acetyl-CoA synthetase has been partially purified and characterized; it is essential for growth only on ethanol, malonate and acetate plus glyoxylate, as shown by the growth characteristics of a mutant ( 1~~5 4 ) lacking this enzyme. Free acetate is not involved in the assimilation of acetyl-CoA, and hydroxypyruvate reductase is not involved in the oxidation of acetyl-CoA to glyoxylate during growth on 3-hydroxybutyrate. A mutant ( I C T~ I ) , lacking ' malate synthase' activity, has been isolated and its characteristics indicate that this activity is normally essential for growth of Pseudomonas A M I on ethanol, malonate and 3-hydroxybutyrate, but not for growth on other substrates such as pyruvate, succinate and C, compounds. The growth properties of a revertant ( I C T~I R ) and of a mutant lacking malyl-CoA lyase ( P C T~~) indicate that an alternative route must exist for assimilation of compounds metabolized exclusively by way of acetyl-CoA.

Cloning, mutagenesis, and physiological effect of a hydroxypyruvate reductase gene from Methylobacterium extorquens AM1

“…We have also presented information suggesting that 20BL contains at least two separate lesions, one affecting HPR and one affecting C-2 metabolism. This putative second lesion, however, could not be the same as those in mutants PCT48 and C5, which have been shown to be defective in the glycine synthesis pathway (8,27), since, unlike PCT48 and C5, 20BL can grow well on pyruvate (our results) and hydroxybutyrate (30). It is also possible that 20BL contains a single lesion that affects HPR as well as enzymes involved in glycine synthesis and that our inability to complement 20BL reflects a lack of the appropriate clone in our clone bank.…”

“…Their growth on both methanol and ethanol was restored in the presence of either glyoxylate or glycollate (6,8,26,27). Since 20BL has been the only available HPR mutant in M. extorquens AM1, this phenotype has resulted in confusion concerning the assimilation of C-2 compounds (8,30). Our data show that the product of hprA, which is clearly the major HPR in M. extorquens AM1, is not required for growth on C-2 compounds.…”

“…The net assimilation of C-1 compounds into cell constituents by this pathway requires the regeneration of one molecule of glycine for each molecule of 3-phosphoglycerate formed. The mechanism of synthesis of glycine from C-1 compounds remains unknown, but it is known that acetyl coenzyme A (acetyl-CoA) is a precursor (30). The use of labeled compounds and mutant studies have shown that the unknown route involved in the synthesis of glycine from acetyl-CoA is also essential for growth on ethanol, pyruvate, lactate, malonate, and hydroxybutyrate (6-8, 26, 27, 30).…”

“…The M. extorquens AM1 methanol mutant 20BL was generated by nitrosoguanidine mutagenesis and lacks hydroxypyruvate reductase (HPR) (12). It does not grow on ethanol, but it grows normally on hydroxybutyrate (8,30). These data suggested that this key enzyme of the serine cycle might play a second role in the route for conversion of acetyl-CoA into glycine during growth on C-2 compounds (8) but not during growth on hydroxybutyrate (30).…”

“…It does not grow on ethanol, but it grows normally on hydroxybutyrate (8,30). These data suggested that this key enzyme of the serine cycle might play a second role in the route for conversion of acetyl-CoA into glycine during growth on C-2 compounds (8) but not during growth on hydroxybutyrate (30). However, the genetic lesion in 20BL was not determined, and so it was not clear whether the phenotype observed was due to a single mutation or whether the structural gene for the serine cycle HPR was affected.…”