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| Hauptseite > Publikationsdatenbank > Biosynthese von Phosphonaten: Charakterisierung des rekombinanten Enzyms Phosphonopyruvat-Decarboxylase aus $\textit{Streptomyces viridochromogenes}$ Tü494 |
| Hauptseite > Publikationsdatenbank > Biosynthese von Phosphonaten: Charakterisierung des rekombinanten Enzyms Phosphonopyruvat-Decarboxylase aus $\textit{Streptomyces viridochromogenes}$ Tü494 |
| Dissertation / PhD Thesis/Book | PreJuSER-45865 |
2005
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
ISBN: 3-89336-400-5
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Please use a persistent id in citations: http://hdl.handle.net/2128/2460
Abstract: Phosphonopyruvate (PPyr), the physiological substrate of the phosphonopyruvate decarboxylase (Ppd), belongs to the phosphonates, which are characterised by a stable, covalent carbon to phosphorus (C-P) bond that makes these compounds resistant against chemical hydrolysis, thermal decomposition, and photolysis. Phosphonates are used as antibiotics, herbicides and in different fields of human economic activity. Furthermore, they are used as anti-metabolites or as inhibitors for the determination of enzyme crystal structures. Their biosynthesis starts from phosphoenolpyruvate (PEP) and is mostly initiated by the enzyme combination PEP phosphomutase (Ppm) and Ppd. The Ppd, that catalyses the decarboxylation of PPyr to phosphonoacetaldehyde (PnAA), causes the elimination of PPyr from the Ppm-catalysed PEP to PPyr rearrangement and therefore the establishment of the C-P bond. The aim of this work was to investigate the biochemical characteristics as well as the correlation between structure and function of the Ppd from $\textit{S. viridochromogenes}$ Tü494, whose activity depends on thiamine pyrophosphate (TPP) and Me$^{2+}$ ions. Thus, suitable assay systems were required. Another purpose was the chemo-enzymatic synthesis of new C-P compounds that could be used as inhibitory molecules (e.g., to resolve enzyme crystal structures). The $\textit{ppd}$ gene was expressed in a recombinant $\textit{E. coli}$ strain as a fusion protein with an aminoterminal 10-fold histidine-tag and purified to homogeneity by Ni$^{2+}$-affinity chromatography. PnAA synthesis was verified by two coupled enzyme activity assays. The genes from $\textit{Pseudomonas putida}$ KT2440, which encode the 2-aminoethylphosphonate transaminase (Aept) and the PnAA hydrolase (Phtase), as well as the Ppm-encoding gene from $\textit{Mytilus edulis}$ were expressed and purified as histidine fusion proteins, too. In combination with alcohol dehydrogenase (ADH), L-lactate dehydrogenase (LDH), and pyruvate kinase four different assay reactions were established. The in vitro synthesis of 2-aminoethylphosphonate was carried out by the combined use of the fusion proteins Ppm, Ppd, and Aept. The pH rate profile followed a bell-shaped curve and defined pH 7.5 – 8.5 as the range for optimal Ppd catalysis. A half-life of about 50 days was determined for storage at -20°C or 4°C. The Ppd has a high affinity to its cofactors TPP and Mg$^{2+}$ (K$_{m}$ ~ 40 μM). The metal ions Ca$^{2+}$ and Mn$^{2+}$ were further activators. Using the enzyme combinations Phtase/ADH and Aept/LDH at pH 8.0 (HEPES buffer system) and a temperature of 30°C, the Ppd followed Michaelis-Menten kinetics with PPyr, yielding a K$_{m}$ value of 3.2 $\pm$ 0.35 μM and a v$_{max}$ value of 0.81 $\pm$ 0.01 U/mg. Contrary to PPyr, neither pyruvic acid, nor $\beta$-hydroxypyruvic acid and $\beta$-fluoropyruvic acid were converted to the respective aldehydes. The supposed carboligase activity of the Ppd, which would lead to the synthesis of phosphonoacyloins, could not be demonstrated. Gel filtration chromatography indicated a molecular mass of 72,100 $\pm$ 220. Regarding the subunit size of about 44,000 Da, the quaternary structure of the Ppd appears to be homodimeric. A comparison with the pyruvate decarboxylase (Pdc) from $\textit{Zymomonas mobilis}$ allowed the identification of several amino acid residues, whose potential functions were examined by sitedirected mutagenesis. Considering the kinetic data of these Ppd mutant proteins as well as the crystal structures of the TPP-dependent enzymes Pdc and benzoylformate decarboxylase, a model of the active site was generated. According to that, glutamate-48 seems to be responsible for TPP activation. The residues serine-25, histidine-110, and aspartate-297 affect the catalytic activity and are probably located in the direct vicinity of the substrate binding site. The residues aspartate-265, asparagine-293, and glycine-294 were found within the TPP-binding motif and mark the binding site of the Me2+ ion that is responsible for the anchoring of the TPP molecule. Likely, aspartate-263, being conserved in the sequences of PPyr and sulfopyruvate (SPyr) decarboxylases, appears to be part of the metal ion binding site. Glutamate-224, another amino acid conserved within the PPyr and SPyr decarboxylase sequences, is probably a residue essential for catalytic activity. The assumed roles for glutamate-224 and aspartate-263 were contradictory to the results, which had been described for the corresponding amino acid residues of the Ppd from $\textit{Bacteroides fragilis}$ (Zhang, G. et al. (2003) $\textit{J. Biol. Chem}$. 278: 41302 – 41308).+
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