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Peptide deformylase is an essential Fe2+ metalloenzyme that catalyzes the removal of the N-terminal formyl group from nascent polypeptides in eubacteria. In vivo, the deformylase is capable of deformylating most of the polypeptides in a bacterial cell, which contain diverse N-terminal sequences. In this work, we have developed a combinatorial method to systematically examine the sequence specificity of peptide deformylase. A peptide library that contains all possible N-terminally formylated tetrapeptides was constructed on TentaGel resin, with a unique peptide sequence on each resin bead. Limited treatment with the Escherichia coli deformylase resulted in the deformylation of those peptides that are the most potent substrates of the enzyme. By using an enzyme-linked assay, the beads containing the deformylated peptides were identified and isolated. Peptide sequence analysis using matrix-assisted laser desorption ionization mass spectrometry revealed a consensus sequence, formyl-Met-X-Z-Tyr (X = any amino acid except for aspartate and glutamate; Z = lysine, arginine, tyrosine, or phenylalanine), for the E. coli enzyme. The deformylase is also capable of efficient deformylation of formyl-Phe-Tyr-(Phe/Tyr) peptides. These results demonstrate that, despite being a broad-specificity enzyme, the peptide deformylase deformylates different peptides at drastically different rates. In addition, the selectivity of peptide deformylase for the N-formyl over the N-acetyl group has been studied with N-alpha-fluoroacetyl peptides, and the results suggest that both electronic and steric factors are responsible for the observed specificity. The deformylase was also shown to exhibit esterase activity. These results will facilitate the design of specific deformylase inhibitors as potential antibacterial agents. This combinatorial method should be generally applicable to the study of the substrate specificity of other acylases and peptidases.

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Bmp4 is a downstream gene of Msx1 in early mouse tooth development. In this study, we introduced the Msx1-Bmp4 transgenic allele to the Msx1 mutants in which tooth development is arrested at the bud stage in an effort of rescuing Msx1 mutant tooth phenotype in vivo. Ectopic expression of a Bmp4 transgene driven by the mouse Msx1promoter in the dental mesenchyme restored the expression of Lef-1 and Dlx2 but neither Fgf3 nor syndecan-1 in the Msx1 mutant molar tooth germ. The mutant phenotype of molar but not incisor could be partially rescued to progress to the cap stage. The Msx1-Bmp4 transgene was also able to rescue the alveolar processes and the neonatal lethality of the Msx1 mutants. In contrast, overexpression of Bmp4 in the wild type molar mesenchyme down-regulated Shh and Bmp2 expression in the enamel knot, the putative signaling center for tooth patterning, but did not produce a tooth phenotype. These results indicate that Bmp4 can bypass Msx1 function to partially rescue molar tooth development in vivo, and to support alveolar process formation. Expression of Shh and Bmp2 in the enamel knot may not represent critical signals for tooth patterning.

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