Knots in polypeptide chains have been found in very few proteins. Only two proteins are considered to have a shallow `trefoil' knot, which tucks a few residues at one end of the chain through a loop exposed on the protein surface. Recently, another protein was found by a mathematical algorithm to have a deep `figure‐of‐eight' knot which had not been visually identified. In the present study, the crystal structure of a hypothetical RNA 2′‐O‐ribose methyltransferase from Thermus thermophilus (RrmA) was determined at 2.4 Å resolution and a deep trefoil knot was found for the first time. The present knot is formed by the threading of a 44‐residue polypeptide chain through a 41‐residue loop and is better defined than the previously reported knots. Two of the three catalytic residues conserved in the 2′‐O‐ribose methyltransferase family are located in the knotting loop and in the knotted carboxy‐terminal chain, which is the first observation that the enzyme active site is constructed right on the knot. On the other hand, the amino‐terminal domain exhibits a geometrical similarity to the ribosomal proteins which recognize an internal loop of RNA.

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, CIavibacter 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 B2y type, possessing both D-and L-DAB. The type strains of all the subspecies of CIavibacter michiganensis have D-and L-DAB in almost equal proportions in their cell wall peptidoglycan as previously reported. In contrast, the type strains of CIavibacter 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 165 rDNA sequences and menaquinone profiles. On the basis of these data, the transfer of CIavibacter 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.Research (RIKEN)r I

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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 ␣ 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, Asn 105 -Val-Ile-Val-CysSer-Leu-Cys-Ser-Cys-Thr-Ala-Trp-Pro-Ile-Leu-Gly-LeuPro-Pro-Thr-Trp-Tyr-Lys 128 . 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 ␣Ile 107 to ␣Trp 117 . Furthermore, by using mass spectrometry, protein sequence, and amino acid composition analyses, we have shown that the 112th Cys residue of the ␣ subunit is post-translationally oxidized to a cysteine-sulfinic acid (Cys-SO 2 H) 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.Nitrile hydratase (NHase; EC 4.2.1.84) 1 is a bacterial metalloenzyme catalyzing the hydration of nitriles to corresponding amides (1, 2). NHase consists of two kinds of subunits (␣ and ␤ with the molecular mass values of 23 kDa) and contains non-heme iron (3) or non-corrinoid cobalt (4) atoms. The NHase from Rhodococcus sp. N-771, which is a ␣␤ heterodimer containing a non-heme iron, is inactivated by aerobic incubation in the dark for a half-day (dark-inactivation), whereas the enzyme purified from the dark-inactivated cells is immediately converted to the active form by light irradiation (photoactivation) (5, 6). Recently, it has been revealed that dark-inactivation and photoactivation are controlled by association and photodissociation of nitric oxide (NO) with the non-heme iron center (7-9). Similar photosensitivity is observed in the NHases from Rhodococcus sp. N-774 (10) and R312.2 These NHases seem to be identical to the one from Rhodococcus sp. N-771 because of the same nucleotide sequences (11, 12). 3The iron-containing NHase is the first enzyme with a mononuclear low-spin non-heme iron(III) which is thought to be involved in the catalysis (3). The structure of the iron center in the active form has been studied by various spectroscopies including ESR (3), resonance Raman (13), extended x-ray absorption fine structure (13) and electron nuclear double resonance (14), and the ligand-donor set of N 3 OS 2 has been proposed (14), which is supported by model complexes of the iron center (15-17). Recently, the metal site structure has been studied in detail by means of electro...

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