Characterization of Enzymes Involved in the Synthesis and Processing of Ether Lipids in Archaea and Bacteria

In Archaea, ether lipids play an essential role as the main building blocks of the cellular membrane. Recently, ether lipids have also been discovered in the domain of Bacteria, and the key enzymes that catalyze their synthesis, glycerol 1-phosphate dehydrogenase (AraM) and heptaprenylglyceryl phosphate synthase (HepGPS), have been described. In Bacillales, heptaprenylglyceryl phosphate (HepGP) does not become linked to a second polyprenyl moiety like for ether lipids in Archaea, but is dephosphorylated and acetylated. The enzymes that catalyze these reactions have been identified and characterized whithin this work. The phosphatase PhoB acting on HepGP was enriched from a Bacillus subtilis (B. subtilis) cell extract and identified by mass spectrometry. The B. subtilis gene phoB was amplified and heterologously expressed. The dephosphorylation activity of PhoB could be verified in vitro. Nevertheless, the results indicate that any other phosphatase might catalyze dephosphorylation of HepGP as well, because those enzymes only exhibit a low subtrate specificity. By screening a B. subtilis knockout library for deficiency in acetylation, the yvoF gene product was identified to be the acetyltransferase. The yvoF gene was heterologously expressed and YvoF was characterized biochemically. Its acetyl-CoA dependent activity was verified in vitro and catalytic parameters were obtained by a 5,5´-dithiobis-(2-nitrobenzoic acid)-coupled activity assay and a discontinuous radiometric assay. A membrane assoziation of YvoF was inferred from ultracentrifugation experiments. The in vitro acetyltransferase activity was also verified for another YvoF representative, namely from Bacillus anthracis. Because YvoF is a close relative to maltose O-acetyltransferase (MAT), the activity of YvoF was compared to MAT. YvoF and MAT partially overlap in substrate and product range in vitro, but MAT is not able to complement the yvoF knockout in vivo. It remains to be clarified how this in vivo specialization is achieved, and one answer might be that the cellular localization of the two enzymes is different, as the YvoF substrate HepG is associated to the cellular membrane. The biological function of acetylated ether lipids in B. subtilis is still unknown. However, the identification of two enzymes involved in their biosynthesis pathway brings us closer to answer this intriguing question. The HepGPS belongs to the family of geranylgeranylglyceryl phosphate synthases (GGGPS) and previous studies showed that the members of this family can be dimers or hexamers. Hexameric GGGPS complexes are built from three dimeric modules in the configuration of the native dimers. Besides the dimeric interface (symmetric interface 1), two additional interfaces exist in the hexamer, the symmetric interface 2 and the asymmetric interface 3a/3b. To elucidate the impact of hexamerization on stability and activity, the GGGPS from Methanothermobacter thermautotrophicus (mtGGGPS) was investigated by mutational studies. The symmetric interface 2 and the asymmetric interface 3a/3b were shown to contribute equally to the stabilization of the hexameric structure, because both led to dimerization if disturbed. A mutation that disrupts the dimeric interface led to a further collapse of the dimers into monomers. The thermal stability of the resulting monomeric, dimeric and hexameric mtGGGPS mutants was determined via various biophysical techniques (circular dichroism-, differential scanning calorimetry-, nano differential scanning fluorimetry-measurements and irreversible heat inactivation). In almost all cases, a higher oligomerization state ensured an elevated thermostability and contributed to maintaining activity at higher temperatures. Based on these results, a putative unfolding pathway for mtGGGPS was proposed. First, a transition of the native structure to a stable partially folded intermediate occurs, which is inactive but maintains almost 80-90 % of the overall secondary structure. Depending on the oligomerization state, this 1st transition happens at a temperature between 50 °C (monomeric mutants), 60 °C (dimeric mutants) and 100 °C (hexameric wild type and mutants). Only at elevated temperatures of around 120 °C a second transition presumably leads to a complete denaturation of the protein, irrespective of the oligomerization state. Data obtained with two other couples of hexameric GGGPS wild type and dimeric mutant as well as the analysis of native hexameric and dimeric GGGPS enzymes from different species confirmed the results obtained with mtGGGPS. A steady-state kinetic experiment revealed that the dimeric mutants showed decreased catalytic efficiencies especially due to an increase of the KM for G1P. In summary, it could be shown for almost all hexameric enzymes that when compared to dimeric or monomeric enzymes, hexamerization ensured structural integrity as a prerequisite for thermal stability and activity.