Biol. Chem., Vol. 391, pp. 1419–1428, December 2010 • Copyright  by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2010.130 2010/220 Article in press - uncorrected proof Kinetic and structural characterization of bacterial glutaminyl cyclases from Zymomonas mobilis and Myxococcus xanthus David Ruiz Carrillo 1,2,a , Christoph Parthier 2,a , Nadine Ja ¨nckel 1 , Julia Grandke 1 , Marco Stelter 1,2 , Stephan Schilling 1 , Mathias Boehme 1 , Piotr Neumann 2 , Raik Wolf 1 , Hans-Ulrich Demuth 1, *, Milton T. Stubbs 2, * and Jens-Ulrich Rahfeld 1 1 Probiodrug AG, Weinbergweg 22, D-06120 Halle/Saale, Germany 2 Institut fu ¨r Biochemie und Biotechnologie, Martin-Luther- Universita ¨t Halle-Wittenberg, Kurt-Mothes-Straße 3, D-06120 Halle/Saale, Germany * Corresponding authors e-mail: Hans-Ulrich.Demuth@probiodrug.de; stubbs@biochemtech.uni-halle.de Abstract Although enzymes responsible for the cyclization of amino- terminal glutamine residues are present in both plant and mammal species, none have yet been characterized in bac- teria. Based on low sequence homologies to plant glutaminyl cyclases (QCs), we cloned the coding sequences of putative microbial QCs from Zymomonas mobilis (ZmQC) and Myxo- coccus xanthus (MxQC). The two recombinant enzymes exhibited distinct QC activity, with specificity constants k cat /K m of 1.47 "0.33 mM -1 s -1 (ZmQC) and 142 "32.7 mM -1 s -1 (MxQC) towards the fluorescent substrate gluta- mine-7-amino-4-methyl-coumarine. The measured pH-rate profile of the second order rate constant displayed an inter- esting deviation towards the acidic limb of the pH chart in the case of ZmQC, whereas MxQC showed maximum activ- ity in the mild alkaline pH range. Analysis of the enzyme variants ZmQCGlu 46 Gln and MxQCGln 46 Glu show that the exchanged residues play a significant role in the pH behav- iour of the respective enzymes. In addition, we determined the three dimensional crystal structures of both enzymes. The tertiary structure is defined by a five-bladed b-propeller anchored by a core cation. The structures corroborate the putative location of the active site and confirm the proposed relation between bacterial and plant glutaminyl cyclases. Keywords: bacterial; glutaminyl cyclase; microbial; pH-rate dependency; pyroglutamate; QC. Introduction Protein function can be adapted by a wide variety of post- translational modifications. In this context, the cyclization of These authors contributed equally to this work. a N-terminal glutamine residues leading to pyroglutamate (pGlu, see Scheme 1) has attracted growing attention. Although glutamine cyclization can occur spontaneously (Busby et al., 1987; Arri et al., 1999), the reaction is cata- lyzed in biological systems by the action of glutaminyl cyclases (QCs, EC 2.3.2.5). The existence of these enzymes was first unveiled in the mid-1960s following isolation from the latex of the plant Carica papaya (Messer, 1963), fol- lowed later by detection of a corresponding mammalian QC activity in bovine pituitary (Busby et al., 1987; Fischer and Spiess, 1987) and isolation of the responsible mammalian enzyme (Pohl et al., 1991). Plant QCs act extracellularly, as demonstrated by their presence in the secreted latex of Carica papaya (Azarkan et al., 2004). Similarly, the cDNA sequences of the mammalian QCs possess signal sequences (Pohl et al., 1991) that are expected to target the protein to the secretory granules asso- ciated with the endoplasmic reticulum (Fischer and Spiess, 1987). In mammals, formation of amino-terminal pGlu has been observed in hormones such as gastrin, neurotensin, GnRH and TRH (Pohl et al., 1991; Awade et al., 1994) and in chemokines such as CCL2 (Proost et al., 1996). In addi- tion, amyloidogenic peptides, e.g., b-amyloid peptide, have been implicated as putative substrates of QC enzymes, whereby an N-terminal glutamate is transformed instead of glutamine (Schilling et al., 2004). The regulation of pGlu- containing amyloidotic peptide homeostasis has been hypothesized to play a key role in the exacerbated neuro- toxicity, stability and aggregation tendencies of such b-amy- loid peptides (Saido et al., 1995; Iwatsubo et al., 1996). By contrast, the biological function of the plant QCs is still unclear. Although plant QCs might also play a role in peptide maturation, involvement in wound healing and defence mechanisms has also been suggested (Azarkan et al., 2004). The mammalian and plant glutaminyl cyclases provide an example of convergent evolution: the two families share the same enzymatic activity yet exhibit neither DNA sequence (Pohl et al., 1991; Dahl et al., 2000) nor three-dimensional structural homology (Huang et al., 2005; Wintjens et al., 2006). Whereas the plant Carica papaya QC (CpQC) dis- plays a five-fold b-propeller structure (Guevara et al., 2006; Wintjens et al., 2006), the human enzyme adopts an a/b hydrolase tertiary organization (Huang et al., 2005). The existence of microbial QCs has been suggested based on sequence homology to the plant enzymes (Pohl et al., 1991; Dahl et al., 2000), but this has not as yet been demonstrated experimentally. Here we present enzymatic and structural evidence for the presence of microbial QCs (mQC) in two Gram-negative bacteria. 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