The 2.8 A resolution structure of Streptomyces griseus protease B and its homology with alpha-chymotrypsin and Streptomyces griseus protease A.

The 2.8 A (1 A = 0.1 nm) resolution structure of the crystalline orthorhombic form of the microbial serine protease Streptomyces griseus protease B (SGPB) has been solved by the method of multiple isomorphous replacement using five heavy-atom derivatives. The geometrical arrangement of the active site quartet, Ser-214, Asp-102, His-57, and Ser-195, is similar to that found for pancreatic alpha-chymotrypsin. SGPB and alpha-chymotrypsin have only 18% identity of primary structure but their tertiary structures are 63% topologically equivalent within a root mean square deviation of 2.07 A. The major tertiary structural differences between the bacterial enzyme SGPB and the pancreatic enzymes is due to the zymogen requirement of the multicellular organisms in order to protect themselves against autolytic degradation. The two pronase enzymes, SGPB and Streptomyces griseus protease A (SGPA), have 61% identity of sequence and their tertiary structures are 85% topologically equivalent within a root mean square deviation of 1.46 A. The active site regions of SGPA and SGPB are similar and their tertiary structures differ only in three minor regions of surface loops.     

Maleylation and pH-dependence of Streptomyces griseus protease B.

The enzymatic activity of Streptomyces griseus protease B (SGPB) was measured over pH range 8.4--11.5 using a specific new, chromophoric substrate N-succinyl-glycyl-glycyl-L-phenylalanine p-nitroanilide. It was found that the activity is dependent on ionization of a single group with apparent pK = 10.84, possibly lysine-125. Maleylation of the epsilon-amino group of this lysine was linearily associated with the loss of enzymatic activity. It is therefore suggested that the electrostatic interaction between the side chain of lysine-125 and the alpha-carboxyl group of the C-terminal tyrosine is crucial to the active conformation of the enzyme. In contrast the maleylation of the alpha-amino group of the N-terminal isoleucine was rapid but could not be correlated to the loss of activity.     

The primary structure of the glutamic acid-specific protease of Streptomyces griseus

The amino acid sequence and part of the DNA sequence of a glutamic acid-specific serine protease from Streptomyces griseus is reported. This protease is shown to be homologous with other serine proteases. An improved purification protocol for this enzyme is described.     

Protein structure refinement: Streptomyces griseus serine protease A at 1.8 A resolution.

The crystal structure of the bacterial serine protease from Streptomyces griseus (SGPA) has been refined at 1.8 Å resolution by a restrained parameter least-squares procedure (Konnert, 1976) to a conventional R factor of 0.139 for 12662 statistically significant reflections [I > 3σ(I)]. The number of variable parameters in the final model was 5912 which included positional and individual thermal parameters of the enzyme, and positions, B factors and occupancies of 175 solvent molecules. The algorithm used for this refinement allows for the simultaneous restraint on bond distances and distances related to interbond angles, the coplanarity of atoms in planar groups, the conservation of chirality of asymmetric centres, non-bonded contact distances, conformational torsional angles and individual isotropic temperature factors.The refined structure of SGPA differs from ideal bond lengths by an overall root-mean-square deviation of 0.02 Å; the corresponding value for angle distances is 0.038 Å. Comparison of the phase angles for the shell of data, 8.0 to 2.8 Å, between the multiple isomorphous replacement phases (Brayer et al., 1978a) and the refined phases, indicates an overall difference (r.m.s.) of 56.6 °. The average conformational angle of the peptide bond (ω) is 179.7 ° (root-mean-square deviation ± 2.5 °) for the 180 peptide bonds of SGPA. Of the 175 solvent molecules included during the course of the refinement, 22 with occupancies ranging from 1.00 to 0.38 are located in the active site and the substrate binding region. It was not until these water molecules were included in the refinement process that the active Ser195 adopted its final conformation (χ¹ = ?77 °). The resulting distance from O^γ of Ser195 to N^ε2 of His57 is 3.1 Å, which, when taken with the observed distortion from linearity (50 °), indicates a rather weak interaction.     

Properties of Streptomyces griseus aminopeptidase

The paper deals with studying the properties of aminopeptidase isolated from Str. griseus culture fluid. The preparation is characterized by a high specific activity and heat stability, it has no admixtures of carboxypeptidases and proteinases. The enzyme is easily inhibited by EDTA, but the addition of Ca2+ evokes its complete reactivation. A partial recovery of the activity may be also reached under the influence of some other bivalent metals. In hydrolysis of di- and tripeptides it is shown that the enzyme has a preferential effect on the substrates with N-terminal leucine. Peptides with N-terminal alanine, valine and glycine are almost not hydrolyzed. The use of the native insulin and decapeptide with the known amino acidic sequence as substrates shows that aminopeptidase can hydrolyze proteins and peptides with the successive release of some amino acids: phenylalanine, serine triptophane, valine, asparagine, etc. Glycine is difficult for removal and may inhibit the further hydrolysis of the polypeptide chain.     

Chromogenesis mirabilis in Streptomyces griseus

A number of chromogenic Streptomyces, producing diffusible melanoid pigment on complex organic media, fail to form melanin pigment on conventionally used synthetic tyrosine agar. By means of our new melanin formation test, almost all the chromogenic streptomyces can now be detected in chemically defined medium. In contrast to ordinary chromogenic streptomyces, two streptomyces species of the International Streptomyces Project, S. griseus ISP 5236 and S. ornatus ISP 5307, produce melanin pigment only on synthetic tyrosine agar, without showing chromogenicity on complex organic media. From the results obtained with S. griseus ISP 5236 and S. phaeochromogenes ISP 5073, it was revealed that melanin formation by Streptomyces, in general, is inhibited by L-cysteine present in organic nitrogen sources incorporated into natural media. Most chromogenic species of streptomyces produce a higher level of tyrosinase and rapidly utilize L-cysteine in the culture media which result in the manifestation of good chromogenicity on natural media. Peculiarity of chromogenicity of S. griseus and S. ornatus might be due to the lower ability to produce tyrosinase and to utilize L-cysteine in the culture medium.     

L-Asparaginase Production by Streptomyces griseus

Streptomyces griseus ATCC 10137 synthesizes about 1 IU of L-asparaginase/100 ml of a 4% peptone medium. The enzyme has a pH optimum of 8.5 which is comparable to that of the L-asparaginase derived from Escherichia coli which has antitumor properties.