Taxonomic significance of 2,4-diaminobutyric acid isomers in the cell wall peptidoglycan of actinomycetes and reclassification of Clavibacter toxicus as Rathayibacter toxicus comb. nov.

An HPLC procedure which separates D- and L-amino acid isomers was applied to an analysis of peptidoglycan of 2,4-diaminobutyric acid (DAB)-containing actinomycetes. The cell wall peptidoglycans of species of the genera Agromyces, Clavibacter and Rathayibacter contain DAB and have been differentiated principally by their menaquinone profile. These peptidoglycans are known to be identical in structure, all being of the B2 gamma type, possessing both D- and L-DAB. The type strains of all the subspecies of Clavibacter michiganesis have D- and L-DAB in almost equal proportions in their cell wall peptidoglycan as previously reported. In contrast, the type strains of Clavibacter toxicus and all valid species of the genera Agromyces and Rathayibacter contain the L-isomer of DAB almost exclusively. This characteristic is in good agreement with phylogenetic analyses based on 16S rDNA sequences and menaquinone profiles. On the basis of these data, the transfer of Clavibacter toxicus to the genus Rathayibacter as Rathayibacter toxicus comb. nov. is proposed. The isomer profile of DAB is shown to be a good taxonomic marker to differentiate these genera.

Structure of the photoreactive iron center of the nitrile hydratase from Rhodococcus sp. N-771. Evidence of a novel post-translational modification in the cysteine ligand.

Nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated by nitrosylation of the non-heme iron center and activated by photodissociation of nitric oxide (NO). To obtain structural information on the iron center, we isolated peptide complexes containing the iron center by proteolysis. When the tryptic digest of the alpha subunit isolated from the inactive form was analyzed by reversed-phase high performance liquid chromatography, the absorbance characteristic of the nitrosylated iron center was observed in the peptide fragment, Asn105-Val-Ile-Val-Cys-Ser-Leu-Cys-Ser-Cys-Thr-Ala-Trp-Pro-Ile-Leu - Gly-Leu-Pro-Pro-Thr-Trp-Tyr-Lys128. The peptide contained 0.79 mol of iron/mol of molecule as well as endogenous NO. Subsequently, by digesting the peptide with thermolysin, carboxypeptidase Y, and leucine aminopeptidase M, we found that the minimum peptide segment required for the nitrosylated iron center is the 11 amino acid residues from alphaIle107 to alphaTrp117. Furthermore, by using mass spectrometry, protein sequence, and amino acid composition analyses, we have shown that the 112th Cys residue of the alpha subunit is post-translationally oxidized to a cysteine-sulfinic acid (Cys-SO2H) in the NHase. These results indicate that the NHase from Rhodococcus sp. N-771 has a novel non-heme iron enzyme containing a cysteine-sulfinic acid in the iron center. Possible ligand residues of the iron center are discussed.

Isolation and primary structure of a novel pheromonotropic neuropeptide structurally related to leucopyrokinin from the armyworm larvae, Pseudaletia separata.

A novel pheromonotropic neuropeptide has been isolated from a head extract of the armyworm larvae, Pseudaletia separata, by a seven step purification procedure using an in vivo assay with decapitated female moths of Bombyx mori. Amino acid sequence analysis and comparison with synthetic peptides established the primary structure of the peptide, termed Pseudaletia pheromonotropin (Pss PT), as H-Lys-Leu-Ser-Tyr-Asp-Asp-Lys-Val-Phe-Glu-Asn-Val-Glu-Phe-Thr-Pro-Arg-Le u-NH2. Pss PT is structurally related to leucopyrokinin, an insect myotropic neuropeptide, and possesses the C-terminal pentapeptide, Phe-Thr-Pro-Arg-Leu-NH2, responsible for the biological activity.