Function of the conserved triad residues in the class C β‐lactamase from Citrobacter freundii GN346

Abstract

The conserved KTG triad in the class C /I-lactamase from Cirrohucter,freundii GN346 was examined as to its function by means of site-directed mutagencsls.The following conversions were performed; Lys-3 15 to arginine, alanine or glutamic acid, Thr-3 I6 to valine, and Gly-3 I7 to alanine, proline or isoleucine. The resultant mutant enzymes revealed that a basic amino acid at position 315 and a small uncharged residue at position 317 are essential for the enzyme activity, but a hydroxyl group at residue 316 is not required for the enzymatic catalysis. The kinetic properties of the purified Arg-3 15 and Val-3 16 enzymes provided information on the function of these residues.

Effects of the Val211Gly substitution on molecular dynamics of the CMY‐2 cephalosporinase: Implications on hydrolysis of expanded‐spectrum cephalosporins

“…The catalytic apparatus of cephalosporinases is unique among serine hydrolases in that it lacks a pure charge‐relay system like the one found in chymotrypsin (Ser 95 ‐His 57 ‐Asp 102 ) or in class A β‐lactamases (Ser 70 ‐Lys 73 ‐Glu 166 ) to activate the nucleophiles during acylation and deacylation. The double displacement in AmpCs is performed by Ser64, Lys67, and Tyr15038–40 in ways that are not yet fully understood. Acyl‐transfer of β‐lactam to Ser64 could occur via proton transfer from serine's hydroxyl to Lys67 through Tyr15041, with the alternative way Ser64→Lys67→Tyr150→leaving‐nitrogen to be also possible 33, 42.…”

Role of β‐lactam carboxyl group on binding of penicillins and cephalosporins to class C β‐lactamases

“…As shown above, the hydroxyl group of Thr316 in the native–penicillin G complex interacts with the O 2 atom in the β‐lactam carboxyl group (I interaction) in penicillins but not in cephalosporins. The loss of the hydroxyl group at position 316 (or 235) by the effect of the Thr316Ala,21 Thr316Val,36 and Ser235Ala32, 34, 35 mutations prevents the enzyme from establishing an I interaction. On the basis of the above‐described results, however, this need not be crucial in either case because the O 2 atom in penicillins can still interact with the enzyme via the Asn346 residue (K interaction), whereas that in cephalosporins never interacts in this way (I interaction).…”

“…Ser130, Lys234, Ser235, and Ala237) (Fig. 1) have revealed marked changes in the hydrolytic activity of penicillins and cephalosporins, particularly on replacement of the residues at positions 316 and 318 in class C β‐lactamases or their analogs at 235 and 237 (316 of 235 and 318 of 237) in class A β‐lactamases 18, 22, 24–36. Because these residues play no direct role in the hydrolysis mechanism, their significance lies in molecular recognition and in retention of the three‐dimensional structure of the active site.…”

A survey of a functional amino acid of class Cβ‐lactamase corresponding to Glu166 of class A β‐lactamases

“…Lys6' and Lys31s of the GN346 enzyme were also coned to be the counte~arts of LYS'~ and Lys'", respectively, by means of site-directed mutagenesis. When these two basic amino acids in the GN346 enzyme were substituted with glutamic acid, the enzyme completely lost the catalytic activity [12,14]. GIu'~~ in class A enzymes was assumed to act as the general base in the acylation or deacylation reaction [8,33].…”