Structure-Function Analysis of Escherichia coli MnmG (GidA), a Highly Conserved tRNA-Modifying Enzyme

ABSTRACT The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-Å resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.

An NH2-terminal deleted plasma membrane H+-ATPase is a dominant negative mutant and is sequestered in endoplasmic reticulum derived structures

The NH2-terminus of the plasma membrane H+-ATPase is one of the least conserved segments of this protein among fungi. We constructed and expressed a mutant H+-ATPase from Saccharomyces cerevisiae deleted at an internal peptide within the cytoplasmic NH2-terminus (D44-F116). When the enzyme was subjected to limited trypsinolysis it was digested more rapidly than wild type H+-ATPase. Membrane fractionation experiments and immunofluorescence microscopy, using antibodies against H+-ATPase showed that the mutant ATPase is retained in the endoplasmic reticulum. The pattern observed in the immunofluorescence microscopy resembled structures similar to Russell bodies (modifications of the endoplasmic reticulum membranes) recently described in yeast. When the wild type H+-ATPase was co-expressed with the mutant, wild type H+-ATPase was also retained in the endoplasmic reticulum. Co-expression of both ATPases in a wild type yeast strain was lethal, demonstrating that this is a dominant negative mutant.

Studies on the structural and functional aspects of Rhodotorula gracilisd-amino acid oxidase by limited trypsinolysis

The structure-function relationships of purified Rhodotorula gracilis D-amino acid oxidase (in its holo-, apo- and holo-enzyme-benzoate complex forms) was analysed by digestion with trypsin. In all cases trypsin cleaves this 80 kDa dimeric enzyme at the C-terminal region, since the peptide bonds sensitive to proteinase attack are clustered in this region. Digestion of native enzyme with trypsin produced a nicked and truncated form of 38.3 kDa containing two polypeptides of 34 and 5 kDa starting from Met1 and Ala319 respectively, and with detachment of the Thr306-Arg318 and Glu365-Leu368 peptides. Our results show that this *#x2018;core’, folded into a compact structure, is catalytically competent. The acquisition of this nicked form was marked by a shift from a dimeric to a monomeric active enzyme, a result never previously obtained. The deleted sequences, Thr306-Arg318 and Glu365-Leu368, are essential for the monomer-monomer interaction, and, in particular, the region encompassing Thr306-Arg318 should play an essential role in the dimerization process. interestingly, the Ser308-Lys321 sequence present in the lost peptide corresponds to a sequence not present in other known D-amino acid oxidases [Faotto, Pollegioni, Ceciliani, Ronchi and Pilone (1995) Biotechnol. Lett. 17, 193-198]. A role of the cleaved-off region for the thermostabilization of the enzyme is also discussed.