On the dd-Carboxypeptidase Enzyme System of Streptomyces Strain K15

Abstract

Streptomyces KIS possesses a set of exocellular and cell-bound D-alanyl-D-alanine carboxypeptidases. Four of them have been isolated to the stage where each enzyme preparation contains one single penicillin-binding protein. The exocellular 54000-M, enzyme is extremely sensitive to benzylpenicillin and performs low transpeptidase activity on the carbonyl-donor/amino-acceptor tetrapeptide AcLLys(G1y)-DAla-DAla. The exocellular 40000-M, enzyme and the two lysozyme-releasable 40000-M, and 38000-M, enzymes are moderately sensitive to benzylpenicillin and have a high propensity to catalyse dimer formation from the aforementioned tetrapeptide monomerThe D-alanyl-D-alanine carboxypeptidases are enzymes involved in the crosslinking step of bacterial cell wall synthesis [l]. They catalyse attack, by a suitable exogenous nucleophile, of the carbonyl carbon of the DAla-DAla amide bond of LXaa-DAIa-DAia-terminated peptides, where Xaa is most often a diamino acid residue. These enzymes exhibit widely varying molecular weights, sensitivities to fl-lactam antibiotics, specificity profiles for the carbonyl-donor peptides and abilities to use amino compounds as nucleophilic acceptors of the LXaa-DAla moieties in transpeptidation reactions [2]. In addition, they can operate by at least two different mechanisms : four serine DD-carboxypeptidases [3 -51 and one Zn2+ DD-carboxypeptidase [6,7] are known. A survey, carried out under standard but arbitrary conditions, of the activities performed by the culture filtrates obtained from various strains of actinomycetes, led to the isolation of the very highly penicillin-sensitive, 53 OOO-M, R39 DD-carboxypeptidase (from Actinomudura R39) [4], the moderately penicillin-sensitive, 38 OOO-M, R61 DD-carboxypeptidase (from Streptomyces R61) [3], and the highly penicillin-resistant 18 000-M,G DDcarboxypeptidase (from Streptomyces ulbus G) [6-71. The R61 and R39 enzymes are serine peptidases; they are able to perform transpeptidation reactions. The G enzyme is a Zn2+ peptidase; it functions only as a hydrolase. The question whether the R61, R39 and G enzymes were strain-specific or not has been examined. It has been found that Streptomyces strain K1S excretes and/or possesses, bound to its cell envelope, a set of DD-carboxypeptidases. On the basis of their penicillin sensitivities and substrate requirements, one of them is a R39-like enzyme and the others are R61-like enzymes. MATERIALS AND METHODS on-Curboxypeptidase Transpeptidase AssaysThe procedures used were those described previously [8,9]. Unless otherwise stated, the substrate concentrations were : 1.8 mM AczLLys-DAla-DAla (Du-carboxypeptidase activity ; reaction catalysed: AczLLys-uAla-DAla + H2O -+ DAla ~~ Ahhreviutions. AcMur, N-acetylniuramic acid; A2pm, meso-2,2'-diaminopimelic acid; AcLys, Nu-acetyllysine; Hepes, 4-(2-hydroxyethyl)-1 -piperazineethanesulphonic acid.

A putative low-molecular-mass penicillin-binding protein (PBP) of Mycobacterium smegmatis exhibits prominent physiological characteristics of dd-carboxypeptidase and beta-lactamase

“…The high k cat and low K m values indicated a higher k cat /K m ratio and strong affinity of the enzyme for both the artificial and pentapeptide substrates (Table 3) Ghuysen, 1991;Leyh-Bouille et al, 1981;Nguyen-Distèche et al, 1982). The high k cat /K m ratio of MSMEG_2433 with both peptide substrates made its DD-CPase activity comparable to most PBPs that are recognized as conventional DD-CPases (Chowdhury et al, 2012).…”