Neutron and X-ray crystallographic analysis of Achromobacter protease I at pD 8.0: protonation states and hydration structure in the free-form.

The structure of the free-form of Achromobacter protease I (API) at pD 8.0 was refined by simultaneous use of single crystal X-ray and neutron diffraction data sets to investigate the protonation states of key catalytic residues of the serine protease. Occupancy refinement of the catalytic triad in the active site of API free-form showed that ca. 30% of the imidazole ring of H57 and ca. 70% of the hydroxyl group of S194 were deuterated. This observation indicates that a major fraction of S194 is protonated in the absence of a substrate. The protonation state of the catalytic triad in API was compared with the bovine ß-trypsin-BPTI complex. The comparison led to the hypothesis that close contact of a substrate with S194 could lower the acidity of its hydroxyl group, thereby allowing H57 to extract the hydrogen from the hydroxyl group of S194. H210, which is a residue specific to API, does not form a hydrogen bond with the catalytic triad residue D113. Instead, H210 forms a hydrogen bond network with S176, H177 and a water molecule. The close proximity of the bulky, hydrophobic residue W169 may protect this hydrogen bond network, and this protection may stabilize the function of API over a wide pH range.

Characterization of N-deoxyribosyltransferase from Lactococcus lactis subsp. lactis.

A nucleoside N-deoxyribosyltransferase-homologous gene was detected by homological search in the genomic DNA of Lactococcus lactis subsp. lactis. The gene yejD is composed of 477 nucleotides encoding 159 amino acids with only 25% identity, which is low in comparison to the amino acid sequences of the N-deoxyribosyltransferases from other lactic acid bacteria, i.e. Lactobacillus leichmannii and Lactobacillus helveticus. The residues responsible for catalytic and substrate-binding sites in known enzymes are conserved at Gln49, Asp73, Asp93 (or Asp95), and Glu101, respectively. The recombinant YejD expressed in Escherichia coli shows a 2-deoxyribosyl transfer activity to and from both bases of purine and pyrimidine, showing that YejD should be categorized as a class II N-deoxyribosyltransferase. Interestingly, the base-exchange activity as well as the heat stability of YejD was enhanced by the presence of monovalent cations such as K(+), NH(4)(+), and Rb(+), indicating that the Lactococcus enzyme is a K(+)-activated Type II enzyme. However, divalent cations including Mg(2+) and Ca(2+) significantly inhibit the activity. Whether or not the yejD gene product actually participates in the nucleoside salvage pathway of Lc. lactis remains unclear, but the lactic acid bacterium possesses the gene coding for the nucleoside N-deoxyribosyltransferase activated by K(+) on its genome.

Purification, characterization, and gene analysis of cellulase (Cel8A) from Lysobacter sp. IB-9374.

An enzyme that has both beta-1,4-glucanase and chitosanase activities was found in the culture medium of the soil bacterium Lysobacter sp. IB-9374, a high lysyl endopeptidase-producing strain. The enzyme was purified to homogeneity from the culture filtrate using five purification steps and designated Cel8A. The purified Cel8A had a molecular mass of 41 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A pH optimum of 5.0 was found for the beta-1,4-glucanase activity, and pH optima of 5.0 and 7.0 were found for the chitosanase activity. Nucleotide sequencing of the Cel8A gene yielded a deduced amino acid sequence that comprises a 33-amino acid, N-terminal signal peptide and a mature enzyme consisting of a 381-residue polypeptide with a predicted molecular mass of 41,241 Da. The amino acid sequence of the Cel8A, which contains the catalytic module of glycosyl hydrolase family 8, is homologous to beta-1,3-1,4-D-glucanase from Bacillus circulans WL-12 and endoglucanase N-257 from B. circulans KSM-N257.

A second lysine-specific serine protease from Lysobacter sp. strain IB-9374.

A second lysyl endopeptidase gene (lepB) was found immediately upstream of the previously isolated lepA gene encoding a highly active lysyl endopeptidase in Lysobacter genomic DNA. The lepB gene consists of 2,034 nucleotides coding for a protein of 678 amino acids. Amino acid sequence alignment between the lepA and lepB gene products (LepA and LepB) revealed that the LepB precursor protein is composed of a prepeptide (20 amino acids [aa]), a propeptide (184 aa), a mature enzyme (274 aa), and a C-terminal extension peptide (200 aa). The mature enzyme region exhibited 72% sequence identity to its LepA counterpart and conserved all essential amino acids constituting the catalytic triad and the primary determining site for lysine specificity. The lepB gene encoding the propeptide and mature-enzyme portions was overexpressed in Escherichia coli, and the inclusion body produced generated active LepB through appropriate refolding and processing. The purified enzyme, a mature 274-aa lysine-specific endopeptidase, was less active and more sensitive to both temperature and denaturation with urea, guanidine hydrochloride, or sodium dodecyl sulfate than LepA. LepA-based modeling implies that LepB can fold into essentially the same three-dimensional structure as LepA by placing a peptide segment, composed of several inserted amino acids found only in LepB, outside the molecule and that the Tyr169 side chain occupies the site in which the indole ring of Trp169, a built-in modulator for unique peptidase functions of LepA, resides. The results suggest that LepB is an isozyme of LepA and probably has a tertiary structure quite similar to it.

Purification, bacteriolytic activity, and specificity of beta-lytic protease from Lysobacter sp. IB-9374.

Lysobacter sp. IB-9374, which was isolated from soil as a high lysyl endopeptidase-producing strain (Chohnanet al., FEMS Microbiol. Lett., 213, 13-20, 2002), was found to produce a beta-lytic protease capable of lysing gram-positive bacteria such as Staphylococcus aureus, Microccocuseus, and Bacillus subtilis. The Lysobacter strain secreted the beta-lytic protease into the culture medium at a 2.4-fold higher level than Achromobacter lyticus. The enzyme was highly purified through a series of six steps with a high yield. The enzyme was strongly inhibited by tetraethylene-pentamine and 1,10-phenanthroline. The purified enzyme lysed more efficiently almost all the gram-positive bacteria tested than lysozyme, lysostaphin, and mutanolysin. The enzyme was very similar to Achromobacter beta-lytic protease containing one zinc atom in terms of amino acid composition and N-terminal sequence. The nucleotide sequence revealed that the mature enzyme was composed of 179 amino acid residues with additional 198 amino acids at the amino-terminal end of the enzyme. The deduced amino acid sequence of the mature enzyme coincided with that of the Achromobacter enzyme, although the prepro-region showed a 41% sequence identity with the counterpart. These results indicate that Lysobacter sp. is a useful strain for an efficient large-scale preparation of beta-lytic protease capable of lysing bacteria.

Lysobacter strain with high lysyl endopeptidase production.

A new lysyl endopeptidase producing strain, Lysobacter sp. IB-9374, was isolated from soil. This strain secreted the endopeptidase to culture medium at 6-12-fold higher levels relative to Achromobacter lyticus and Lysobacter enzymogenes. The mature Lysobacter sp. enzyme was enzymatically identical to Achromobacter lysyl endopeptidase bearing lysyl bond specificity, a high peptidase activity, a wide pH optimum, and stability against denaturants. Nucleotide sequence analysis of the Lysobacter sp. lysyl endopeptidase gene revealed that the enzyme is synthesized as a precursor protein consisting of signal peptide (20 amino acids (aa)), pro-peptide (185 aa), mature enzyme (268 aa), and C-terminal extension peptide (198 aa). The deduced amino acid sequence of the mature enzyme was totally identical to that of the Achromobacter enzyme. The Lysobacter sp. precursor protein has an 18-aa longer peptide chain following nine consecutive amino acid residues distinct from the Achromobacter counterpart at the C-terminus. Total precursor protein is 671 aa of which only 268 aa are in the finally processed exoenzyme.