Study of the Zn‐containing dd‐carboxypeptidase of Streptomyces albus G by small‐angle X‐ray scattering in solution

Abstract

Study of the Zn'+-containing D-alanyl-D-alanine-cleaving carboxypeptidase of Streptomyces albus G by smallangle X-ray scattering in solution yielded the following molecular parameters: radius of gyration R = 1.82 f 0.05 nm; largest diameter D = 5.9 f 0.2 nm; relative molecular mass M, = 17000 & 2000; volume V zz 35 f 2 nm3; degree of hydration: 0.25 f 0.02 g water/g protein. By reference to theoretical scattering curves of rigid triaxial homogeneous bodies, a model which fits all experimental data is an elliptical cylinder. Such a model is compatible with that observed in the crystal structure. At those high concentrations necessary to form inactive enzyme-ligand associations the non-competitive p-lactam inhibitors, cephalothin and cephalosporin C, drastically altered the scattering behaviour of the protein.The G, R61 and R39 D-alanyl-D-alanine-cleaving peptidases (in short DD-peptidases), isolated from Streptomyces albus G, Streptomyces R61 and Actinomadura R39 respectively, have been used extensively as model enzymes for the study of the reactions involved in the last stages of bacterial wall peptidoglycan synthesis and the mode of action of the p-lactam antibiotics [l]. The R61 and R39 enzymes perform catalysis via an active serine residue and are respectively very and exceedingly sensitive to penicillin. In contrast, the G enzyme operates via a Zn2+ cofactor and is highly resistant to penicillin. Knowledge of the exact atomic level structures and functioning of these enzymes is also relevant to the proposed peptidoglycan network models [2,3]. The G and R61 DD-peptidases have been crystallized. The solution of the R61 enzyme structure has proceeded to a resolution of 0.28 nm, showing, via difference Fourier maps, the binding site of cephalosporin C and 6,6-dichloro-4-deaza-2,2-didemethylpenicillanic acid [4, 51. The amino acid sequence (212 residues) [6] and the threedimensional structure at 0.45nm and 0.25 nm resolution of the G Zn2+ DD-peptidase have been established [7, 81 and a plausible picture of how this enzyme performs catalysis has been proposed [9]. The overall shape of the enzyme in the crystal structure is that of an elliptical cylinder with a height of 4.8nm and an axial ratio of 1.4: 1 :0.8 (height:long axis:short axis). The enzyme conformation in aqueous solution has now been studied by small-angle X-ray scattering. The results obtained are presented here. MATERIALS AND METHODS EnzymeThe enzyme was purified to protein homogeneity [lo]. The enzyme solutions were prepared by extensive dialysis at 4 "C against 50mM Tris/HCl buffer pH8.3 (buffer I) or 10mM Hepes/NaOH buffer pH 7.75 containing 5 mM MgC1, (buffer 11). Enzyme concentrations ranging between about 0.2 mM and 0.8 mM were determined spectrophotometrically at 280 nm, using A ] & = 10. Small-angle X-ray scatteringWeighed amounts of cephalosporin C and cephalothin (from Ely Lilly and Sigma respectively) were added to the enzyme solutions followed by rapid agitation. The molar ratios enzyme: antibiotic were 1 :20 for cephalosporin C in buffer...

Human Peptidoglycan Recognition Proteins Require Zinc to Kill Both Gram-Positive and Gram-Negative Bacteria and Are Synergistic with Antibacterial Peptides

“…Proper function of host immunity requires Zn 2ϩ because Zn 2ϩ -deficient individuals have increased susceptibility to infections and Zn 2ϩ supplementation restores or enhances resistance to infections and the functions of innate and acquired immunity, although the exact mechanisms involved are mostly unknown (34 -40). Several peptidoglycan-or polysaccharide-lytic enzymes contain Zn 2ϩ (43)(44)(45)(46), and some antimicrobial peptides require Zn 2ϩ for their antimicrobial activity (47-52). However, the role of Zn 2ϩ and other divalent cations in innate immunity is complex because, in addition to its enhancing effect, Zn 2ϩ can also inhibit the activity of some antimicrobial proteins, especially Ca 2ϩ -binding proteins psoriasin (26) and calprotectin (27)(28)(29).…”

Active-site-directed inactivators of the Zn2+-containing d-alanyl-d-alanine-cleaving carboxypeptidase of Streptomyces albus G

“…Other f,-lactams (cephalosporin C and cephalothin) are weak non-competitive inhibitors. In parallel to this, these compounds destroy the protein crystal lattice and profoundly alter the scattering behaviour of the protein by inducing large conformational changes and/or aggregation (Labischinski et al, 1984). 6-,B-Iodopenicillanate is an unusual penam in that it has a very short side chain.…”