Mutagenesis and biochemical studies on AuaA confirmed the importance of the two conserved aspartate-rich motifs and sugg
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ORIGINAL PAPER
Mutagenesis and biochemical studies on AuaA conWrmed the importance of the two conserved aspartate-rich motifs and suggested diVerence in the amino acids for substrate binding in membrane-bound prenyltransferases Edyta Stec · Shu-Ming Li
Received: 30 October 2011 / Revised: 16 December 2011 / Accepted: 23 January 2012 / Published online: 7 February 2012 © Springer-Verlag 2012
Abstract AuaA is a membrane-bound farnesyltransferase from the myxobacterium Stigmatella aurantiaca involved in the biosynthesis of aurachins. Like other known membrane-bound aromatic prenyltransferases, AuaA contains two conserved aspartate-rich motifs. Several amino acids in the Wrst motif NXxxDxxxD were proposed to be responsible for prenyl diphosphate binding via metal ions like Mg2+. Site-directed mutagenesis experiments demonstrated in this study that asparagine, but not the arginine residue in NRxxDxxxD, is important for the enzyme activity of AuaA, diVering from the importance of NQ or ND residues in the NQxxDxxxD or NDxxDxxxD motifs observed in some membrane-bound prenyltransferases. The second motif of known membrane-bound prenyltransferases was proposed to be involved in the binding of their aromatic substrates. KDIxDxEGD, also found in AuaA, had been previously speculated to be characteristic for binding of Xavonoids or homogenisate. Site-directed mutagenesis experiments with AuaA showed that KDIxDxEGD was critical for the enzyme activity. However, this motif is very likely not speciWc for Xavonoid or homogenisate prenyltransferases, because none of the tested Xavonoids was accepted by AuaA or its mutant R53A in the presence of farnesyl, geranyl or dimethylallyl diphosphate.
Communicated by Erko Stackebrandt. Electronic supplementary material The online version of this article (doi:10.1007/s00203-012-0795-0) contains supplementary material, which is available to authorized users. E. Stec · S.-M. Li (&) Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, 35037 Marburg, Germany e-mail: [email protected]
Keywords Aurachin biosynthesis · Farnesyltransferase · Membrane-bound prenyltransferase · Myxobacteria · Mutagenesis
Introduction Aromatic prenyltransferases (PTs) catalyze the transfer of a prenyl moiety from a prenyl diphosphate onto an aromatic substrate and can be classiWed, based on their sequences and biochemical properties, in at least three groups (Brandt et al. 2009; Heide 2009). One group consisting of soluble enzymes like CloQ (Pojer et al. 2003) or NphB (Kuzuyama et al. 2005) is mainly found in bacteria, but also in fungi (Haug-SchiVerdecker et al. 2010). These PTs do not posses conserved aspartate-rich motifs and their catalytic activities were dependent (Kuzuyama et al. 2005) or independent (Pojer et al. 2003) of the presence of metal ions. Structural analysis of NphB and CloQ showed that several basic amino acid residues are involved in the coordination of prenyl diphosphate moiety (Kuzuyama et al. 2005; Metzger et al. 2010). Th
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