Cyclodipeptide synthases (CDPSs) constitute a family of peptide bond-forming enzymes that use aminoacyl-tRNAs for the synthesis of cyclodipeptides. Here, we describe the activity of 41 new CDPSs. We also show that CDPSs can be classified into two main phylogenetically distinct subfamilies characterized by specific functional subsequence signatures, named NYH and XYP. All 11 previously characterized CDPSs belong to the NYH subfamily, suggesting that further special features may be yet to be discovered in the other subfamily. CDPSs synthesize a large diversity of cyclodipeptides made up of 17 proteinogenic amino acids. The identification of several CDPSs having the same specificity led us to determine specificity sequence motifs that, in combination with the phylogenetic distribution of CDPSs, provide a first step toward being able to predict the cyclodipeptides synthesized by newly discovered CDPSs. The determination of the activity of ten more CDPSs with predicted functions constitutes a first experimental validation of this predictive approach.
The glpK genes of Enterococcus casseliflavus and Enterococcus faecalis, encoding glycerol kinase, the key enzyme of glycerol uptake and metabolism in bacteria, have been cloned and sequenced. The translated amino acid sequences exhibit strong homology to the amino acid sequences of other bacterial glycerol kinases. After expression of the enterococcal glpK genes in Escherichia coli, both glycerol kinases were purified and were found to be phosphorylated by enzyme I and the histidine-containing protein of the phosphoenolpyruvate: glycose phosphotransferase system. Phosphoenolpyruvate-dependent phosphorylation caused a 9-fold increase in enzyme activity. The site of phosphorylation in glycerol kinase of E. casseliflavus was determined as His-232. Site-specific mutagenesis was used to replace His-232 in glycerol kinase of E. casseliflavus with an alanyl, glutamate, or arginyl residue. The mutant proteins could no longer be phosphorylated confirming that His-232 of E. casseliflavus glycerol kinase represents the site of phosphorylation. The His 232 3 Arg glycerol kinase exhibited an about 3-fold elevated activity compared with wild-type glycerol kinase. Fructose 1,6-bisphosphate was found to inhibit E. casseliflavus glycerol kinase activity. However, neither EIIA Glc from E. coli nor the EIIA Glc domain of Bacillus subtilis had an inhibitory effect on glycerol kinase of E. casseliflavus.Glycerol uptake in Gram-negative and Gram-positive bacteria is mediated by the glycerol diffusion facilitator, an integral membrane protein of 30 kDa, catalyzing the rapid equilibration of concentration gradients of glycerol across the cytoplasmic membrane (1, 2). Intracellular glycerol is converted to glycerol-3-P by the enzyme glycerol kinase that uses ATP as phosphoryl donor (3, 4). Glycerol-3-P is not a substrate of the glycerol diffusion facilitator (5) and hence remains entrapped in the cell, where it is further metabolized. Phosphorylation of glycerol by glycerol kinase provides the driving force for the uptake of glycerol, as it creates a constant imbalance of the glycerol concentrations inside and outside the cell. It was therefore not surprising that glycerol kinase is the target of several regulation mechanisms.
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