Crystal structure of the alkaline proteinase Savinase from Bacillus lentus at 1.4 A resolution.

Savinase (EC3.4.21.14) is secreted by the alkalophilic bacterium Bacillus lentus and is a representative of that subgroup of subtilisin enzymes with maximum stability in the pH range 7 to 10 and high activity in the range 8 to 12. It is therefore of major industrial importance for use in detergents. The crystal structure of the native form of Savinase has been refined using X-ray diffraction data to 1.4 A resolution. The starting model was that of subtilisin Carlsberg. A comparison to the structures of the closely related subtilisins Carlsberg and BPN' and to the more distant thermitase and proteinase K is presented. The structure of Savinase is very similar to those of homologous Bacillus subtilisins. There are two calcium ions in the structure, equivalent to the strong and the weak calcium-binding sites in subtilisin Carlsberg and subtilisin BPN', well known for their stabilizing effect on the subtilisins. The structure of Savinase shows novel features that can be related to its stability and activity. The relatively high number of salt bridges in Savinase is likely to contribute to its high thermal stability. The non-conservative substitutions and deletions in the hydrophobic binding pocket S1 result in the most significant structural differences from the other subtilisins. The different composition of the S1 binding loop as well as the more hydrophobic character of the substrate-binding region probably contribute to the alkaline activity profile of the enzyme. The model of Savinase contains 1880 protein atoms, 159 water molecules and two calcium ions. The crystallographic R-factor [formula; see text].     

Calcium-mediated thermostability in the subtilisin superfamily: the crystal structure of Bacillus Ak.1 protease at 1.8 A resolution

Proteins of the subtilisin superfamily (subtilases) are widely distributed through many living species, where they perform a variety of processing functions. They are also used extensively in industry. In many of these enzymes, bound calcium ions play a key role in protecting against autolysis and thermal denaturation. We have determined the crystal structure of a highly thermostable protease from Bacillus sp. Ak.1 that is strongly stabilized by calcium. The crystal structure, determined at 1.8 A resolution (R=0. 182, Rfree=0.247), reveals the presence of four bound cations, three Ca(2+) and one Na(+). Two of the Ca(2+) binding sites, Ca-1 and Ca-2, correspond to sites also found in thermitase and the mesophilic subtilisins. The third calcium ion, however, is at a novel site that is created by two key amino acid substitutions near Ca-1, and has not been observed in any other subtilase. This site, acting cooperatively with Ca-1, appears to give substantially enhanced thermostability, compared with thermitase. Comparisons with the mesophilic subtilisins also point to the importance of aromatic clusters, reduced hydrophobic surface and constrained N and C termini in enhancing the thermostability of thermitase and Ak.1 protease. The Ak.1 protease also contains an unusual Cys-X-Cys disulfide bridge that modifies the active site cleft geometry.     

The refined crystal structure of subtilisin Carlsberg at 2.5 A resolution

We report here the X-ray crystal structure of native subtilisin Carlsberg, solved at 2.5 A resolution by molecular replacement and refined by restrained least squares to a crystallographic residual (Formula see text): of 0.206. we compare this structure to the crystal structure of subtilisin BPN'. We find that, despite 82 amino acid substitutions and one deletion in subtilisin Carlsberg relative to subtilisin BPN', the structures of these enzymes are remarkably similar. We calculate an r.m.s. difference between equivalent alpha-carbon positions in subtilisin Carlsberg and subtilisin BPN' of only 0.55 A. This confirms previous reports of extensive structural homology between these two subtilisins based on X-ray crystal structures of the complex of eglin-c with subtilisin Carlsberg [McPhalen, C.A., Schnebli, H.P. and James, M.N.G. (1985) FEBS Lett., 188, 55; Bode, W., Papamokos, E. and Musil, D. (1987) Eur. J. Biochem., 166, 673-692]. In addition, we find that the native active sites of subtilisins Carlsberg and BPN' are virtually identical. While conservative substitutions at residues 217 and 156 may have subtle effects on the environments of substrate-binding sites S1' and S1 respectively, we find no obvious structural correlate for reports that subtilisins Carlsberg and BPN' differ in their recognition of model substrates. In particular, we find no evidence that the hydrophobic binding pocket S1 in subtilisin Carlsberg is 'deeper', 'narrower' or 'less polar' than the corresponding binding site in subtilisin BPN'.     

Crystal structure of an alkaline protease from Bacillus alcalophilus at 2.4 A resolution

The crystal structure of an alkaline protease from Bacillus alcalophilus has been determined by X-ray diffraction at 2.4 A resolution. The enzyme crystallizes in space group P2(1)2(1)2(1) with lattice constants a = 53.7, b = 61.6, c = 75.9 A. The structure was solved by molecular replacement using the structure of subtilisin Carlsberg as search model. Refinement using molecular dynamics and restrained least squares methods results in a crystallographic R-factor of 0.185. The tertiary structure is very similar to that of subtilisin Carlsberg. The greatest structural differences occur in loops at the surface of the protein.     

Refined crystal structure of the complex of subtilisin BPN' and Streptomyces subtilisin inhibitor at 1.8 A resolution

The crystal structure of subtilisin BPN' complexed with a proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was refined at 1.8 A resolution to an R-factor of 0.177 with a root-mean-square deviation from ideal bond lengths of 0.014 A. The work finally established that the SSI-subtilisin complex is a Michaelis complex with a distance between the O gamma of active Ser221 and the carbonyl carbon of the scissile peptide bond being an intermediate value between a covalent bond and a van der Waals' contact, 2.7 A. This feature, as well as the geometry of the catalytic triad and the oxyanion hole, is coincident with that found in other highly refined crystal structures of the complex of subtilisin Novo, subtilisin Carlsberg, bovine trypsin or Streptomyces griseus protease B with their proteinaceous inhibitors. The enzyme-inhibitor beta-sheet interaction is composed of two separate parts: that between the P1-P3 residues of SSI and the 125-127 chain segment (the "S1-3 site") of subtilisin and that between the P4-P6 residues of SSI and th 102-104 chain segment (the "S4-6 site") of subtilisin. The latter beta-interaction is unique to subtilisin. In contrast, the beta-sheet interaction previously found in the complex of subtilisin Novo and chymotrypsin inhibitor 2 or in the complex of subtilisin Carlsberg and Eglin C is distinct from the present complex in that the two types of beta-interactions are not separate. As for the flexibility of the molecules comprising the present complex, the following observations were made by comparing the B-factors for free and complexed SSI and comparing those for free and complexed subtilisin BPN'. The rigidification of the component molecules upon complex formation occurs in a very localized region: in SSI, the "primary" and "secondary" contact regions and the flanking region; in subtilisin BPN', the S1-3 and S4-6 sites and the flanking region.     

The 1.4 A crystal structure of the large and cold-active Vibrio sp. alkaline phosphatase

Alkaline phosphatase (AP) from the cold-adapted Vibrio strain G15-21 is among the AP variants with the highest known k(cat) value. Here the structure of the enzyme at 1.4 A resolution is reported and compared to APs from E. coli, human placenta, shrimp and the Antarctic bacterium strain TAB5. The Vibrio AP is a dimer although its monomers are without the long N-terminal helix that embraces the other subunit in many other APs. The long insertion loop, previously noted as a special feature of the Vibrio AP, serves a similar function. The surface does not have the high negative charge density as observed in shrimp AP, but a positively charged patch is observed around the active site that may be favourable for substrate binding. The dimer interface has a similar number of non-covalent interactions as other APs and the "crown"-domain is the largest observed in known APs. Part of it slopes over the catalytic site suggesting that the substrates may be small molecules. The catalytic serines are refined with multiple conformations in both monomers. One of the ligands to the catalytic zinc ion in binding site M1 is directly connected to the crown-domain and is closest to the dimer interface. Subtle movements in metal ligands may help in the release of the product and/or facilitate prior dephosphorylation of the covalent intermediate. Intersubunit interactions may be a major factor for promoting active site geometries that lead to the high catalytic activity of Vibrio AP at low temperatures.     

The structure of neutral protease from Bacillus cereus at 0.2-nm resolution.

The crystal structure of the neutral protease from Bacillus cereus has been refined to an R factor of 17.5% at 0.2-nm resolution. The enzyme, an extracellular metalloendopeptidase, consists of two domains and binds one zinc and four calcium ions. The structure is very similar to that of thermolysin, with which the enzyme shares 73% amino-acid sequence identity. The active-site cleft between the two domains is wider in neutral protease than in thermolysin. This suggests the presence of a flexible hinge region between the two domains, which may assist enzyme action. The high-resolution analysis allows detailed examination of possible causes for the difference in thermostability between neutral protease and thermolysin.     

Crystal structure of thermitase at 1.4 A resolution

The crystal structure of thermitase, a subtilisin-type serine proteinase from Thermoactinomyces vulgaris, was determined by X-ray diffraction at 1.4 A resolution. The structure was solved by a combination of molecular and isomorphous replacement. The starting model was that of subtilisin BPN' from the Protein Data Bank, determined at 2.5 A resolution. The high-resolution refinement was based on data collected using synchrotron radiation with a Fuji image plate as detector. The model of thermitase refined to a conventional R factor of 14.9% and contains 1997 protein atoms, 182 water molecules and two Ca ions. The tertiary structure of thermitase is similar to that of the other subtilisins although there are some significant differences in detail. Comparison with subtilisin BPN' revealed two major structural differences. The N-terminal region in thermitase, which is absent in subtilisin BPN', forms a number of contacts with the tight Ca2+ binding site and indeed provides the very tight binding of the Ca ion. In thermitase the loop of residues 60 to 65 forms an additional (10) beta-strand of the central beta-sheet and the second Ca2+ binding site that has no equivalent in the subtilisin BPN' structure. The observed differences in the Ca2+ binding and the increased number of ionic and aromatic interactions in thermitase are likely sources of the enhanced stability of thermitase.     

1H, 13C and 15N NMR backbone assignments and secondary structure of the 269-residue protease subtilisin 309 from Bacillus lentus

Summary ¹H, ¹³C and ¹⁵N NMR assignments of the backbone atoms of subtilisin 309, secreted by Bacillus lentus, have been made using heteronuclear 3D NMR techniques. With 269 amino acids, this protein is one of the largest proteins to be sequentially assigned by NMR methods to date. Because of the size of the protein, some useful 3D correlation experiments were too insensitive to be used in the procedure. The HNCO, HN(CO)CA, HNCA and HCACO experiments are robust enough to provide most of the expected correlations for a protein of this size. It was necessary to use several experiments to unambiguously determine a majority of the -protons. Combined use of HCACO, HN(COCA)HA, HN(CA)HA, ¹⁵N TOCSY-HMQC and ¹⁵N NOESY-HMQC experiments provided the H chemical shifts. Correlations for glycine protons were absent from most of the spectra. A combination of automated and interactive steps was used in the process, similar to that outlined by Ikura et al. [(1990) J. Am. Chem. Soc., 112, 9020–9022] in the seminal paper on heteronuclear backbone assignment. A major impediment to the linking process was the amount of overlap in the C and H frequencies. Ambiguities resulting from this redundancy were solved primarily by assignment of amino acid type, using C chemical shifts and TOCSY ladders. Ninety-four percent of the backbone resonances are reported for this subtilisin. The secondary structure was analyzed using 3D ¹⁵N NOESY-HMQC data and C secondary chemical shifts. Comparison with the X-ray structure [Betzel et al. (1992) J. Mol. Biol., 223, 427–445] shows no major differences.Supplementary material available from F.J.M. van de Ven: the source code (PASCAL) for the computer program described in this paper.     

The controlled introduction of multiple negative charge at single amino acid sites in subtilisin Bacillus lentus

The use of methanethiosulfonates as thiol-specific modifying reagents in the strategy of combined site-directed mutagenesis and chemical modification allows virtually unlimited opportunities for creating new protein surface environments. As a consequence of our interest in electrostatic manipulation as a means of tailoring enzyme activity and specificity, we have adopted this approach for the controlled incorporation of multiple negative charges at single sites in the representative serine protease, subtilisin Bacillus lentus (SBL). A series of mono-, di- and triacidic acid methanethiosulfonates were synthesized and used to modify cysteine mutants of SBL at positions 62 in the S2 site, 156 and 166 in the S1 site and 217 in the S1' site. Kinetic parameters for these chemically modified mutant (CMM) enzymes were determined at pH 8.6 under conditions which ensured complete ionization of the unnatural amino acid side-chains introduced. The presence of up to three negative charges in the S1, S1' and S2 subsites of SBL resulted in up to 11-fold lowered activity, possibly due to interference with oxyanion stabilization of the transition state of the hydrolytic reactions catalyzed. Each unit increase in negative charge resulted in a raising of K(M) and a reduction of k(cat). However, no upper limit was observed for increases in K(M), whereas decreases in k(cat) reached a limiting value. Comparison with sterically similar but uncharged CMMs revealed that electrostatic effects of negative charges at positions 62, 156 and 217 are detrimental, but are beneficial at position 166. These results indicate that the ground-state binding of SBL to the standard substrate, Suc-AAPF-pNA, to SBL is reduced, but without drastic attenuation of catalytic efficiency, and show that SBL tolerates high levels of charge at single sites.     

X-ray structure determination and comparison of two crystal forms of a variant (Asn115Arg) of the alkaline protease from Bacillus alcalophilus refined at 1.85 A resolution.

The X-ray structure determination, refinement and comparison of two crystal forms of a variant (Asn115Arg) of the alkaline protease from Bacillus alcalophilus is described. Under identical conditions crystals were obtained in the orthorhombic space group P2(1)2(1)2(1) (form I) and the rhombohedral space group R32 (form II). For both space groups the structures of the protease were solved by molecular replacement and refined at 1.85 A resolution. The final R-factors are 17.9% and 17.1% for form I and form II, respectively. The root-mean-square deviation between the two forms is 0.48 A and 0.86 A for main-chain and side-chain atoms, respectively. Due to differences in crystal lattice contacts and packing, the structures of the two crystal forms differ in intermolecular interaction affecting the local conformation of three flexible polypeptide sequences (Ser50-Glu55, Ser99-Gly102, Gly258-Ser259) at the surface of the protein. While the two overall structures are very similar, the differences are significantly larger than the errors inherent in the structure determination. As expected, the differences in the temperature factors in form I and II are correlated with the solvent accessibility of the corresponding amino acid residues. In form II, two symmetry-related substrate binding sites face each other, forming a tight intermolecular interaction. Some residues contributing to this intermolecular interaction are also found to be involved in the formation of the complex between subtilisin Carlsberg and the proteinaceous inhibitor eglin C. This demonstrates that the two symmetry-related molecules interact with each other at the same molecular surface area that is used for binding of substrates and inhibitors.     

Cloning and sequencing of an alkaline protease gene from Bacillus lentus and amplification of the gene on the B. lentus chromosome by an improved technique

A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the protease gene on the chromosome by an improved gene amplification technique.     

Crystal structure at 2.6 A resolution of the complex of subtilisin BPN' with streptomyces subtilisin inhibitor

The crystal structure of the complex of a bacterial alkaline serine proteinase, subtilisin BPN', with its proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was solved at 2.6 A resolution. Compared with other similar complexes involving serine proteinases of the trypsin family, the present structure is unique in several respects. (1) In addition to the usual antiparallel beta-sheet involving the P1, P2 and P3 residues of the inhibitor, the P4, P5 and P6 residues form an antiparallel beta-sheet with a previously unnoticed chain segment (residues 102 through 104, which was named the S4-6 site) of subtilisin BPN'. (2) The S4-6 site does not exist in serine proteinases of the trypsin family, whether of mammalian or microbial origin. (3) Global induced-fit movement seems to occur on SSI: a channel-like structure in SSI where hydrophobic side-chains are sandwiched between two lobes becomes about 2 A wider upon complexing with subtilisin. (4) The complex is most probably a Michaelis complex, as in most of the other complexes. (5) The main role of the "secondary contact region" of SSI seems to be to support the reactive site loop ("primary contact region"). Steric homology of the two contact regions between the inhibitors of the SSI family and the pancreatic secretory trypsin inhibitor-ovomucoid inhibitor family is so high that it seems to indicate divergent evolutionary processes and to support the general notion as to the relationship of prokaryotic and eukaryotic genes put forward by Doolittle (1978).     

A serine alkaline protease from the fungusConidiobolus coronatus with a distinctly different structure than the serine protease subtilisin Carlsberg

In view of the functional similarities between subtilisin Carlsberg and the alkaline protease fromConidiobolus coronatus, the biochemical and structural properties of the two enzymes were compared. In spite of their similar biochemical properties, e.g., pH optima, heat stability, molecular mass, pI, esterase activity, and inhibition by diisopropyl fluorophosphate and phenylmethlysulfonylfluoride, the proteases were structurally dissimilar as revealed by (1) their amino acid compositions, (2) their inhibition by subtilisin inhibitor, (3) their immunological response to specific anti-Conidiobolus protease antibody, and (4) their tryptic peptide maps. Our results demonstrate that although they are functionally analogous, theConidiobolus protease is structurally distinct from subtilisin Carlsberg. TheConidiobolus protease was also different from other bacterial and animal proteases (e.g. pronase, protease K, trypsin, and chymotrypsin) as evidenced by their lack of response to anti-Conidiobolus protease antibody in double diffusion and in neutralization assays. TheConidiobolus serine protease fails to obey the general rule that proteins with similar functions have similar primary sequences and, thus, are evolutionarily related. Our results strengthen the concept of convergent evolution for serine proteases and provide basis for research in evolutionary relationships among fungal, bacterial, and animal proteases.     

Bleach-stable, alkaline protease from Bacillus sp.

A bleach-stable, thermotolerant, alkaline protease for detergent formulation from a newly isolated Bacillus SB5 is reported. Most (85%) activity of the enzyme was retained in the presence of 10% (v/v) H2O2 and 1% SDS (w/v) at 40°C, after 1 h. The enzyme was optimal at pH 10 and 60°C to 70°C. Enzyme activity was enhanced 30 to 80% in presence of ionic and non-ionic detergents, surfactants and commercial detergents or bleach.     

Altering the specificity of subtilisin Bacillus lentus through the introduction of positive charge at single amino acid sites

The use of methanethiosulfonates as thiol-specific modifying reagents in the strategy of combined site-directed mutagenesis and chemical modification allows virtually unlimited opportunities for creating new protein surface environments. As a consequence of our interest in electrostatic manipulation as a means of tailoring enzyme activity and specificity, we have recently adopted this approach for the controlled incorporation of multiple negative charges at single sites in the representative serine protease, subtilisin Bacillus lentus (SBL). We now describe the use of this strategy to introduce multiple positive charges. A series of mono-, di- and triammonium methanethiosulfonates were synthesized and used to modify cysteine mutants of SBL at positions 62 in the S2 site, 156 and 166 in the S1 site and 217 in the S1' site. Kinetic parameters for these chemically modified mutants (CMM) enzymes were determined at pH 8.6. The presence of up to three positive charges in the S1, S1' and S2 subsites of SBL resulted in up to 77-fold lowered activity, possibly due to interference with the histidinium ion formed in the transition state of the hydrolytic reactions catalyzed.     

Crystal structure of the complex of subtilisin BPN' with its protein inhibitor Streptomyces subtilisin inhibitor. The structure at 4.3 Angstroms resolution

The crystal structure of the complex of subtilisin BPN′ (EC 3.4.21.14) with its protein inhibitor (Streptomyces subtilisin inhibitor) was solved at 4.3 Å resolution, thus establishing the following. (1) Two subtilisin BPN′ molecules (2E) associate with one dimeric inhibitor molecule (I₂) to form the complex molecule E₂I₂. (2) The conformation of neither the inhibitor nor subtilisin BPN′ undergoes any detectable change at this resolution upon complex formation. (3) The inhibitor binds to subtilisin to form an antiparallel β-sheet, as in the case of trypsin/ trypsin inhibitor complexes. (4) The scissible bond of the inhibitor is between Met73′ and Val74′, as proposed earlier (Ikenaka et al., 1974). (5) The protein inhibitor and the substrates bind to subtilisin BPN′ in essentially the same way.