D-alanyl-D-alanine carboxypeptidase in the bacterial form and L-form of Proteus mirabilis.

Membranes of the bacterial form and the stable and unstable L-forms of Proteus mirabilis contain LD and DD-carboxypeptidase. The DD-carboxypeptidase is inhibited non-competitively by penicillin G. The enzyme of the bacterial form is highly penicillin-sensitive (Ki - 4 X 10(-9) M penicillin G). Inhibition is only partly reversible by treatment with penicillinase or by dialysis against buffer. In contrast, the DD-carboxypeptidase of the unstable L-form, grown in the presence of penicillin, is 175-fold less penicillin-sensitive (Ki = 7 X 10(7) M penicillin G). Inhibition is completely reversed by penicillinase or dialysis. After inhibition by penicillin and subsequent reactivation the penicillin sensitivity of the bacterial DD-carboxtpeptidase is similar to the sensitivity of the enzyme of the unstable L-form. The hypothesis is proposed that P. mirabilis contains two DD-carboxypeptidases of different penicillin sensitivity and with different mechanisms of penicillin binding. Peptidoglycan synthesis in the cell walls of the unstable L-form is probably carried out with the help of only one DD-carboxypeptidase, viz. the completely reactivatable enzyme with the lower penicillin sensitivity.     

Solubilization and isolation of the membrane-bound DD-carboxypeptidase of Streptococcus faecalis ATCC9790. Properties of the purified enzyme.

Streptococcus faecalis ATCC 9790 possesses six membrane-bound, penicillin-binding proteins. That numbered 6 (Mr 43000) is the most abundant one and is the DD-carboxypeptidase studied previously. The enzyme has been solubilized and purified to the stage where one single protein band can be detected by gel electrophoresis. The purification procedure does not alter the properties that the enzyme exhibits when it is membrane-bound. The DD-carboxypeptidase itself may be a killing target for penicillin in S. faecalis.     

Biosynthesis of peptidoglycan in Gaffkya homari. The mode of action of penicillin G and mecillinam.

The effect of the beta-lactam antibiotics penicillin G and mecillinam on the incorporation of peptidoglycan into pre-formed cell wall peptidoglycan was studied with wall membrane enzyme preparations from Gaffkya homari. Using UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) as precursors the incorporation of peptidoglycan into the pre-existing cell wall of G. homari was inhibited to an extent of 50% (ID50 value) at a concentration of 0.25 mug of penicillin G/ml. With UDP-GlcNAc and UDP-MurNAc-tetrapeptide as precursors the ID50 value was about 2500-fold greater (630 mug/ml). The inhibition by penicillin G of the incorporation of peptidoglycan from UDP-MurNAc-[14C]Lys-pentapeptide could be overcome by addition of non-radioactive UDP-MurNAc-tetrapeptide to the incubation mixture. In the presence of 5 mug of penicillin G/ml the incorporation of peptidoglycan formed from the mixture of UDP-MurNAc-Ala-DGlu-Lys-D-[14C]Ala-D[14C]Ala and non-radioactive UDP-MurNAc-tetrapeptide proceeded virtually without release of D-[14C]alanine by transpeptidase activity. The enzyme preparation also exhibited DD-carboxypeptidase activity which was only slightly more sensitive to penicillin G and mecillinam than was the incorporation of peptidoglycan into the cell wall. Since the ID50 values for the beta-lactam antibiotics are similar to the concentrations required to inhibit the growth of G. homari to an extent of 50%, the DD-carboxypeptidase must be the killing site of both penicillin G and mecillinam.     

Streptomyces R61 DD-carboxypeptidase: hydrolysis of X-D-alanyl-D-alanine peptides measured by a fluorometric assay

A fluorometric procedure for measuring the activity of DD-carboxypeptidase is described. The method is based on the reaction of one of the products, D-alanine, with o-phthaldialdehyde to form a highly fluorescent adduct. The method has been applied in examining a series of X-D-alanyl-D-alanine peptides as substrates of the penicillin-sensitive DD-carboxypeptidase from Streptomyces R61. The effect of the third residue, X, on kinetic parameters and its implications on the steric analog model for penicillin action are also discussed.     

Thermal inactivation of immobilized penicillin acylase in the presence of substrate and products

Inactivation of immobilized penicillin acylase has been studied in the presence of substrate (penicillin G) and products (phenylacetic acid and 6-aminopenicillanic acid), under the hypothesis that substances which interact with the enzyme molecule during catalysis will have an effect on enzyme stability. The kinetics of immobilized penicillin acylase inactivation was a multistage process, decay constants being evaluated for the free-enzyme and enzyme complexes, from whose values modulation factors were determined for the effectors in each enzyme complex at each stage. 6-Aminopenicillanic acid and penicillin G stabilized the enzyme in the first stage of decay. Modulation factors in that stage were 0.96 for penicillin G and 0.98 for 6-aminopenicillanic acid. Phenylacetic acid increased the rate of inactivation in both stages, modulating factors being -2.31 and -2.23, respectively. Modulation factors influence enzyme performance in a reactor and are useful parameters for a proper evaluation. (c) 1996 John Wiley & Sons, Inc.     

Crystal structure of penicillin binding protein 4 (dacB) from Escherichia coli, both in the native form and covalently linked to various antibiotics

The crystal structure of penicillin binding protein 4 (PBP4) from Escherichia coli, which has both DD-endopeptidase and DD-carboxypeptidase activity, is presented. PBP4 is one of 12 penicillin binding proteins in E. coli involved in the synthesis and maintenance of the cell wall. The model contains a penicillin binding domain similar to known structures, but includes a large insertion which folds into domains with unique folds. The structures of the protein covalently attached to five different antibiotics presented here show the active site residues are unmoved compared to the apoprotein, but nearby surface loops and helices are displaced in some cases. The altered geometry of conserved active site residues compared with those of other PBPs suggests a possible cause for the slow deacylation rate of PBP4.     

The crystal structure of a penicilloyl-serine transferase of intermediate penicillin sensitivity. The DD-transpeptidase of streptomyces K15.

The serine DD-transpeptidase/penicillin-binding protein of Streptomyces K15 catalyzes peptide bond formation in a way that mimics the penicillin-sensitive peptide cross-linking reaction involved in bacterial cell wall peptidoglycan assembly. The Streptomyces K15 enzyme is peculiar in that it can be considered as an intermediate between classical penicillin-binding proteins, for which benzylpenicillin is a very efficient inactivator, and the resistant penicillin-binding proteins that have a low penicillin affinity. With its moderate penicillin sensitivity, the Streptomyces K15 DD-transpeptidase would be helpful in the understanding of the structure-activity relationship of this penicillin-recognizing protein superfamily. The structure of the Streptomyces K15 enzyme has been determined by x-ray crystallography at 2.0-A resolution and refined to an R-factor of 18.6%. The fold adopted by this 262-amino acid polypeptide generates a two-domain structure that is close to those of class A beta-lactamases. However, the Streptomyces K15 enzyme has two particular structural features. It lacks the amino-terminal alpha-helix found in the other penicilloyl-serine transferases, and it exhibits, at its surface, an additional four-stranded beta-sheet. These two characteristics might serve to anchor the enzyme in the plasma membrane. The overall topology of the catalytic pocket of the Streptomyces K15 enzyme is also comparable to that of the class A beta-lactamases, except that the Omega-loop, which bears the essential catalytic Glu(166) residue in the class A beta-lactamases, is entirely modified. This loop adopts a conformation similar to those found in the Streptomyces R61 DD-carboxypeptidase and class C beta-lactamases, with no equivalent acidic residue.     

Characterization of the beta-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies

The binding of penicillin to penicillin acylase was studied by X-ray crystallography. The structure of the enzyme-substrate complex was determined after soaking crystals of an inactive betaN241A penicillin acylase mutant with penicillin G. Binding of the substrate induces a conformational change, in which the side chains of alphaF146 and alphaR145 move away from the active site, which allows the enzyme to accommodate penicillin G. In the resulting structure, the beta-lactam binding site is formed by the side chains of alphaF146 and betaF71, which have van der Waals interactions with the thiazolidine ring of penicillin G and the side chain of alphaR145 that is connected to the carboxylate group of the ligand by means of hydrogen bonding via two water molecules. The backbone oxygen of betaQ23 forms a hydrogen bond with the carbonyl oxygen of the phenylacetic acid moiety through a bridging water molecule. Kinetic studies revealed that the site-directed mutants alphaF146Y, alphaF146A and alphaF146L all show significant changes in their interaction with the beta-lactam substrates as compared with the wild type. The alphaF146Y mutant had the same affinity for 6-aminopenicillanic acid as the wild-type enzyme, but was not able to synthesize penicillin G from phenylacetamide and 6-aminopenicillanic acid. The alphaF146L and alphaF146A enzymes had a 3-5-fold decreased affinity for 6-aminopenicillanic acid, but synthesized penicillin G more efficiently than the wild type. The combined results of the structural and kinetic studies show the importance of alphaF146 in the beta-lactam binding site and provide leads for engineering mutants with improved synthetic properties.     

Membrane-bound DD-carboxypeptidase and transpeptidase activities from Bacillus megaterium KM at pH 7. General properties, substrate specificity and inhibition by beta-lactam antibiotics.

1. The membranes from Bacillus megaterium KM contained a DD-carboxypeptidase with optimum activity under the following conditions: pH 7; ionic strength, 1.3 M; temperature, 40 degrees C and below 20 degrees C. It did not require any divalent cation, but was inactivated by Cu2+ and Hg2+. It was stimulated by 2-mercaptoethanol and low concentrations of p-chloromercuribenzoate. 2. The membrane preparation also catalyzed a simple transpeptidation reaction using as carboxyl acceptors D-alanine or glycine. 3. The conditions for optimum activity, temperature-inactivation, temperature-dependence of the activity, carboxyl donor specificity, sensitivity to beta-lactam antibiotics, and insensitivity to potential peptide inhibitors of both enzyme activities, was identical. The DD-carboxypeptidase showed inhibition by D-alanine and Ac2-L-Lys-D-Ala. 4. The inhibition by beta-lactam antibiotic was reversible for both enzymic activities and the time-dependence for their recovery was identical. 5. The DD-carboxypeptidase was very sensitive to changes in the configuration and size of the side-chains of the C-terminal dipeptide of the substrate. Amino acid residues at the C-terminus that precluded the peptide from being a DD-carboxypeptidase substrate were not acceptors in the transpeptidation reaction. Dipeptides were not acceptors for the 'model transpeptidase'. 6. It is suggested that both activities are catalysed by the same enzyme molecule, whose physiological role is not the formation of peptide crosslinks during peptidoglycan biosynthesis.     

Substrate specificity of penicillin acylase from Streptomyces lavendulae

The kinetic parameters of several substrates of penicillin acylase from Streptomyces lavendulae have been determined. The enzyme hydrolyses phenoxymethyl penicillin (penicillin V) and other penicillins with aliphatic acyl-chains such as penicillin F, dihydroF, and K. The best substrate was penicillin K (octanoyl penicillin) with a k(cat)/K(m) of 165.3 mM(-1) s(-1). The enzyme hydrolyses also chromogenic substrates as NIPOAB (2-nitro-5-phenoxyacetamido benzoic acid), NIHAB (2-nitro-5-hexanoylamido benzoic acid) or NIOAB (2-nitro-5-octanoylamido benzoic acid), however failed to hydrolyse phenylacetil penicillin (penicillin G) or NIPAB (2-nitro-5-phenylacetamido benzoic acid) and penicillins with polar substituents in the acyl moiety. These results suggest that the structure of the acyl moiety of the substrate is more determinant than the amino moiety for enzyme specificity. The enzyme was inhibited by several organic acids and the extent of inhibition changed with the hydrophobicity of the acid. The best inhibitor was octanoic acid with a K(i) of 0.8 mM. All the results, taking together, point to an active site highly hydrophobic for this penicillin acylase from Streptomyces lavendulae.     

The peptidoglycan crosslinking enzyme system in Streptomyces strains R61, K15 and rimosus

The DD-carboxypeptidase-transpeptidase enzyme system in Streptomyces strain K15 consists of: (1) a membrane-bound transpeptidase capable of performing low DD-carboxypeptidase activity; and (2) a set of DD-carboxypeptidases: (a) membrane-bound, (b) lysozyme-releasable and (c) exocellular, having low transpeptidase activities in aqueous media and at low acceptor concentrations. The DD-carboxypeptidases are related to each other and may belong to the same pathway leading to enzyme excretion. A similar enzyme system occurs in Streptomyces strain R61 except that the membrane-bound DD-carboxypeptidase activity is low when compared with the membrane-bound transpeptidase activity. In Streptomyces rimosus the enzyme system consists almost exclusively of the membrane-bound transpeptidase and the levels of membrane-bound, lysozyme-releasable and exocellular DD-carboxypeptidases are very low.     

聚丙烯腈纤维固定化青霉素酰化酶性质的研究

将巨大芽孢杆菌（Bacillusmegaterium）青霉素酞化酶连接到聚丙烯腈纤维载体上,制成固定化青霉素酰化酶。其表现活力约为2000u／g。水解青霉素G的最适温度为50℃;最适PH为9．0;在PHS．5～10．3、温度50℃以下酶的活力稳定;表观米氏常数Ka为1．33×10-8mol／L;最大反应速度Vm为2．564mmol·min-1;苯乙酸为竞争性抑制剂,抑制常数为0．16mol／L。水解10％的青霉素G钾盐溶液,使用20批,保留酶活力80％。     

Production of the penicillin precursor δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV) by cell-free extracts from Streptomyces clavuligerus

Abstract Glycerol-stabilised cell extracts of Streptomyces clavuligerus contain an enzyme activity which synthesises ACV from the individual amino acids L -α-aminoadipic acid, L -cysteine and L -valine. Enzyme activity was optimum in reaction mixtures containing 1 mM ATP together with an ATP regenerating system. The ACV synthetase enzyme formed ACV analogs when provided with L - carboxymethylcysteine in place of L -α-aminoadipic acid or when provided with L - allo isoleucine or L -α-aminobutyrate in place of L -valine. Multistep conversion of individual amino acids to penicillin and cephalosporin antibiotics was restricted as a result of the inhibitory effects of L -α-aminoadipic acid and L -cysteine on isopenicillin N synthetase.     

AmpH, a bifunctional DD-endopeptidase and DD-carboxypeptidase of Escherichia coli

In Escherichia coli, low-molecular-mass penicillin-binding proteins (LMM PBPs) are important for correct cell morphogenesis. These enzymes display DD-carboxypeptidase and/or dd-endopeptidase activities associated with maturation and remodeling of peptidoglycan (PG). AmpH has been classified as an AmpH-type class C LMM PBP, a group closely related to AmpC β-lactamases. AmpH has been associated with PG recycling, although its enzymatic activity remained uncharacterized until now. Construction and purification of His-tagged AmpH from E. coli permitted a detailed study of its enzymatic properties. The N-terminal export signal of AmpH is processed, but the protein remains membrane associated. The PBP nature of AmpH was demonstrated by its ability to bind the β-lactams Bocillin FL (a fluorescent penicillin) and cefmetazole. In vitro assays with AmpH and specific muropeptides demonstrated that AmpH is a bifunctional DD-endopeptidase and DD-carboxypeptidase. Indeed, the enzyme cleaved the cross-linked dimers tetrapentapeptide (D45) and tetratetrapeptide (D44) with efficiencies (k(cat)/K(m)) of 1,200 M(-1) s(-1) and 670 M(-1) s(-1), respectively, and removed the terminal D-alanine from muropeptides with a C-terminal D-Ala-D-Ala dipeptide. Both DD-peptidase activities were inhibited by 40 μM cefmetazole. AmpH also displayed a weak β-lactamase activity for nitrocefin of 1.4 × 10(-3) nmol/μg protein/min, 1/1,000 the rate obtained for AmpC under the same conditions. AmpH was also active on purified sacculi, exhibiting the bifunctional character that was seen with pure muropeptides. The wide substrate spectrum of the DD-peptidase activities associated with AmpH supports a role for this protein in PG remodeling or recycling.     

Contribution of membrane-binding and enzymatic domains of penicillin binding protein 5 to maintenance of uniform cellular morphology of Escherichia coli

Four low-molecular-weight penicillin binding proteins (LMW PBPs) of Escherichia coli are closely related and have similar DD-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of E. coli have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other DD-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal beta-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the DD-carboxypeptidase enzymatic core.     

A method for screening penicillin G acylase-producing bacteria by means of 2-nitro-5-phenylacetaminobenzoic acid test paper

A simple, rapid assay for screening penicillin G acylase-producing bacteria is presented. The method is based on the formation of yellow 2-nitro-5-aminobenzoic acid by penicillin G acylase acting on 2-nitro-5-phenylacetaminobenzoic acid (NIPAB). NIPAB test paper is briefly applied to bacterial colonies on the agar surface, which are subsequently scored individually on the paper by color; bright yellow indicates the presence of penicillin G acylase, natural color its absence. The present method is suitable not only for screening penicillin G acylase-production by a variety of bacteria but also for detection from a large number of transformant colonies of clones containing a gene encoding for the enzyme.     

Affinity of exocellular DD-carboxypeptidase/transpeptidase from Saccharopolyspora erythraea PZH TZ-575 to beta-lactam compounds

The DD-carboxypeptidase/transpeptidases (DD-peptidases) involved in bacterial cell wall metabolism, catalyse the attack of C-terminal D-alanyl-D-alanine peptide bond of the peptydoglycan precursor. These enzymes are inactivated by beta-lactam antibiotics. DD-peptidase from Saccharopolyspora erythraea PZH TZ 64-575 was purified by the use of DEAE-cellulose, Sephadex G-100, Q-Sepharose resins and FPLC (Mono Q). After each step the effluent was concentrated by Amicon ultrafiltration. The purified enzyme showed DD-carboxypeptidase specific activity of 50.9 U/mg. The enzyme exhibited high affinity to beta-lactam compounds e.g. cefamandole, cefapirin, cefradin 1.5-2.6 x 10(-8) M. It was used to screen strains from the Culture Collection of the National Institute of Hygiene in Warsaw for the production of DD-peptidase inhibitors.     

Enzyme production by genetically engineered Streptomyces strains: influence of culture conditions

Production of various extracellular enzymes (the beta-lactamases from Streptomyces albus G, Streptomyces cacaoi, Actinomadura R39, and the DD-carboxypeptidase from Streptomyces R61) by genetically engineered Streptomyces lividans TK24 in Lennox broth medium reached a maximum after 36 to 48 h. Subsequently, the enzyme activity drastically decreased probably due to an increased pH value and the production of an inactivator by Streptomyces lividans. Protease activity did not seem to play a major role. The increased pH and inactivator synthesis are related to amino acid catabolism and generally result in cellularlysis. The use of a medium where the catabolism of amino acids was made less likely by the presence of glucose and NH(4)Cl and by buffering at pH 7.4 considerably inproved the yield. Furthermore, the water activity of the medium seemed to be an important parameter for the production of extracellular proteins by genetically engineered Streptomyces. Better production was observed when the water activity was decreased to 0.96-0.98 by addition of sucrose.Under those conditions, the concentration of extracellular enzyme reached about 0.3 g (1 g in the best case)/L of culture supernantant.     

Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase

Deacetoxycephalosporin/deacetylcephalosporin C synthase (DAOC/DACS) is an iron(II) and 2-oxoglutarate-dependent oxygenase involved in the biosynthesis of cephalosporin C in Cephalosporium acremonium. It catalyzes two oxidative reactions, oxidative ring-expansion of penicillin N to deacetoxycephalosporin C, and hydroxylation of the latter to give deacetylcephalosporin C. The enzyme is closely related to deacetoxycephalosporin C synthase (DAOCS) and DACS from Streptomyces clavuligerus, which selectively catalyze ring-expansion or hydroxylation reactions, respectively. In this study, structural models based on DAOCS coupled with site-directed mutagenesis were used to identify residues within DAOC/DACS that are responsible for controlling substrate and reaction selectivity. The M306I mutation abolished hydroxylation of deacetylcephalosporin C, whereas the W82A mutant reduced ring-expansion of penicillin G (an "unnatural" substrate). Truncation of the C terminus of DAOC/DACS to residue 310 (Delta310 mutant) enhanced ring-expansion of penicillin G by approximately 2-fold. A double mutant, Delta310/M306I, selectively catalyzed the ring-expansion reaction and had similar kinetic parameters to the wild-type DAOC/DACS. The Delta310/N305L/M306I triple mutant selectively catalyzed ring-expansion of penicillin G and had improved kinetic parameters (K(m) = 2.00 +/- 0.47 compared with 6.02 +/- 0.97 mm for the wild-type enzyme). This work demonstrates that a single amino acid residue side chain within the DAOC/DACS active site can control whether the enzyme catalyzes ring-expansion, hydroxylation, or both reactions. The catalytic efficiency of mutant enzymes can be improved by combining active site mutations with other modifications including C-terminal truncation and modification of Asn-305.     

Identification of an extracellular nucleotide pyrophosphatase in the culture media of Streptomyces mediterranei ME/R 17]

An exocellular pyrophosphatase, active on the nucleotide precursors of peptidoglycans, has been found in the culture medium of Streptomyces mediterranei ME/R 17. This enzyme was separated from the DD-carboxypeptidase by batchwise adsorption on DEAE cellulose. The pyrophosphatase had no strict substrate requirements, it hydrolyzed various UDP-sugar substrates: UDP-GlcNAc, UDP-Mur NAc and UDP-MurNAc peptides, giving rise to the corresponding sugar phosphate and to UMP. The enzyme preparation also contained a 5'-nucleotidase activity and UMP was further split to give uridine. This nucleotidase activity was inhibited by potassium tetraborate. Both cytoplasmic and particulate preparations from cells of S. mediterranei also contained a pyrophosphatase activity while only the particulate fractions showed the DD-carboxypeptidase activity. The pyrophosphatase excretion was tested during the grwoth cycle. The activity of the enzyme showed a constant increase throughout the exponential growth and a stronger increase in the late exponential phase. Such a result could be correlated with a consumption of the nutrients in the culture medium, in fact a relatively poor culture medium had a strong positive effect upon the production of the exocellular pyrophosphatase.