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Aote: BLA, ^-lactamase; C Base, carboxy peptides; DAP, D-aminopeptidase; PBP, penicillin-binding protein.

Aote: BLA, ^-lactamase; C Base, carboxy peptides; DAP, D-aminopeptidase; PBP, penicillin-binding protein.

toward (D-Phe)3 and (D-Phe)4, forming (D-Phe)2 and D-Phe. The enzyme is also active toward tripeptides with D-Tyr at the C- or N-terminus and on Boc-(D-Phe)n (n = 2-4), forming Boc-D-Phe, (D-Phe)2, and D-Phe. The enzyme had esterase activity toward D-Phe methyl ester and (D-Phe)2 methyl ester. The products from Boc-(D-Phe)3 tert-butyl ester were Boc-D-Phe, D-Phe, and D-Phe tert-butyl ester. The enzyme was not active toward L-Phe methyl ester, (L-Phe)2 methyl ester, (L-Phe)4, Boc-(L-Phe)4, Boc-(L-Phe)4 methyl ester, (D-Val)3, (D-Leu)2, and (D-Ala)n (n = 2-5). These properties indicated that the enzyme is an endopeptidase that acts D-stereospecifically on peptides composed of aromatic D-amino acids. On the other hand, a dimer was formed when D-Phe methyl ester and D-Phe amide were the substrates. Since the enzyme is found to be a serine peptidase as described it is anticipated that later, the enzyme will be useful in the kinetically controlled synthesis of peptides.

Eight stereoisomers of phenylalanine trimer were synthesized, and their effectiveness as substrates for the enzyme was tested. The enzyme recognized the configuration of the second D-Phe of tripeptides and catalyzes the hydrolysis of the second peptide bond from the N-terminus. The calculated Vmax/Km values for the peptides containing L-Phe were lower than that for (D-Phe)3, affected by the neighboring L-Phe. The enzyme also showed /¡-lactamase activity toward ampicillin and penicillin G. The calculated Vmax values of the enzyme for /-lactam compounds were about the same as those for (D-Phe) 3 and (D-Phe) 4, whereas the Km values were several hundred times larger. On the other hand, carboxypeptidase DD (30) and D-aminopepti-dase (28) activities were undetectable.

The time course of the (D-Phe)4 degradation was measured. As shown in Figure 6, (D-Phe)4 was hydrolyzed to (D-Phe) 2 and D-Phe. No (D-Phe)3 was detected. These results coincide with the kinetic properties of the enzyme described earlier. The mode of action of the enzyme was examined with the synthetic substrates D-Tyr-(D-Phe)2 and (D-Phe)2-D-Tyr, as shown in Figure 7. When D-Tyr-(D-Phe)2 was the substrate, D-Phe was released first, then D-Tyr was slowly formed. When (D-Phe)2-D-Tyr was used as a substrate, D-Tyr was released first, then D-Phe was slowly formed. In both reactions, the second peptide bond from the N-terminus of the substrate was hydrolyzed first. These results showed that the enzyme acts as a D-stereo-specific dipeptidylendopeptidase.

The enzyme activity was maximal at 45 °C. About 60% activity remained after an incubation at 43 °C in 0.1 M potassium phosphate buffer, pH 8.0, for 10 min. No activity was lost between pH 5.0 and 10.0 after an incubation at 30 °C for 1 h in 0.05 M buffers at various pH values (48).

The enzyme activity was enhanced by Mg2+ (138%), Mo3+ (130%), and Ba2+ (123%). When measured after incubating at 30 °C for 30 min, the activity was inhibited to 94% by phenylmethylsulfonyl fluoride (PMSF), 76% by Ag + , 74% by Fe2 + , and 32% by Hg2+ at 5 mM. These results, together with the information about its primary structure as described later, indicated that the enzyme is a serine peptidase (48).

Cloning of the Gene for the Alkaline d-Peptidase

The gene coding for alkaline D-peptidase (adp) was cloned into plasmid pUC118, and a 1,164 bp open reading frame consisting of 388 codons with an Mr of 42,033 was identified as the adp gene (Fig. 8) (48). The enzyme would be synthesized with a signal peptide.

The deduced primary structure of the enzyme is similar to carboxypeptidase DD from Streptomyces R61 (35.0% identical over 346 amino acids [a.a.]) (14), penicillin-binding proteins from Streptomyces (Aocardia) lactamdurans (28.1% over 263 a.a.) (49) and that of B. subtilis (28.5% over 309 a.a.) (50), and class C /-lactamases of Serratia marcescens (24.9% over 217 a.a.) (51), class C/-lactamases of Enterobacter cloacae (25.1% over 191 a.a.) (52), fimbrial protein D from Dichelobacternodosus (24.1% over 261 a.a.) (53), D-aminopeptidase from O. anthropi (27.5% over 182 a.a.) (28), and esterase from Pseudomonas sp. (30.5% over 154 a.a.) (54). As shown in Figure 9, the sequence of Ser-Xaa-Xaa-Lys is perfectly conserved among this class of enzymes and the consensus sequence is located around 60 residues from the N-termini of most of the enzymes. Thus, we propose that alkaline D-peptidase from B. cereus be categorized as a new member of the penicillin-recognizing enzymes, which include penicillin-binding proteins, /-lactamases, and D-aminopeptidase. Table 4 summarizes the properties of the D-amino acid-containing peptide hy-drolases, D-aminopeptidase, and alkaline D-peptidase.

Synthesis of d-Phe Oligopeptides by the Alkaline d-Peptidase (55)

The alkaline D-peptidase not only hydrolyzed (D-Phe)3 and (D-Phe)4 to form (D-Phe)2 and D-phenylalanine but also acted on D-Phe methyl ester and D-Phe amide to form (D-Phe)2. This finding gave us the opportunity to use the enzyme to further investigate the synthesis of D-Phe oligo-peptides from D-Phe methyl ester. Because the alkaline D-peptidase was found to be a serine peptidase (48), we attempted to use it for kinetically controlled peptide synthesis. An expression plasmid pKADP was constructed by placing the alkaline D-peptidase gene (adp), amplified by means of the polymerase chain reaction, under the tac promoter of pKK223-3. Oligomerization of D-phenylalanine methyl ester by means of the purified enzyme from the transformant E. coli was investigated under several conditions. D-Phe dimer, (D-Phe)2, and trimer, (D-Phe)3, were produced in 25.4% and 8.6% yield, respectively, when 50 mM of the substrate was incubated for 8 h with ADP (2.0 and 0.4 U/mL, respectively) in 100 mM triethylamine HCl (pH 11.5). Addition of dimethyl sulfoxide to the reaction mixture resulted in the production of tetramer (D-Phe)4 in 6.7% yield with the decrease of the (D-Phe)2 and (D-Phe)3 production. This is the first example of the synthesis of D-phenylalanine oligomers by means of a D-stereospecific en-dopeptidase.

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