A single amino acid substitution in the Ω-like loop of E. coli PBP5 disrupts its ability to maintain cell shape and intrinsic beta-lactam resistance

Abstract

A single amino acid substitution in the V-like loop of E. coli PBP5 disrupts its ability to maintain cell shape and intrinsic beta-lactam resistance PBP5 has an 'V-loop'-like region similar to that in class A beta-lactamases. It was previously predicted that Leu182 present in the 'V-like' loop of PBP5 corresponds to Glu166 in PER-1 beta-lactamase. Here, we studied the physiological and biochemical effects of the Leu182Glu mutation in PBP5. Upon overexpression in septuple PBP mutants,~75 % of cells were abnormally shaped and intrinsic beta-lactam resistance maintenance was partially lost. Biochemically, the purified soluble mutated PBP5 (smPBP5) retained low acylation ability for penicillin. The turnover number of smPBP5 for artificial and peptidoglycan mimetic substrates was~10-fold less than that of the wild-type. Superimposition of the active-site residues of smPBP5 on PBP5 revealed that perturbation in the orientating key residues may explain the low turnover numbers. Therefore, we establish the involvement of Leu182 in maintaining the physiological and biochemical behaviour of E. coli PBP5. INTRODUCTIONOne of the main bacterial cell-wall components is the peptidoglycan layer, which is indispensable for cellular viability and maintenance of cellular morphology. Penicillin-binding proteins (PBPs) are enzymes required for the cross-linking of the glycan chain (transglycosylase) and peptide chain (transpeptidase). Most of these proteins have three signature motifs, namely SXXK, SXN and KTG, and these motifs are responsible for their respective enzymic activities (Ghuysen, 1991). In Escherichia coli, five PBPs (PBP4, 5, 6, DacD and AmpH) are documented as DD-carboxypeptidase (DDCPase) (Korat et al., 1991;Baquero et al., 1996; Höltje, 1998;Nelson & Young 2000, 2001 González-Leiza et al., 2011). These PBPs are thought to prevent the unwanted cross-link formation by removing the terminal D-alanine residue from the pentapeptide side-chain of N-acetyl muramic acid (Höltje, 1998;Ghosh et al., 2008). PBP5 is the key DD-CPase that helps maintain a uniform cell shape in E. coli (Nelson & Young 2000, 2001Ghosh et al., 2008). E. coli mutants lacking at least three DD-CPases including PBP5 show aberrant morphology. However, the abnormalities are abolished upon ectopic expression of PBP5 (Nelson & Young, 2000;Ghosh & Young, 2003). The ability of PBP5 to repair the cellular abnormalities depends on a stretch of 20 aa (200-219) around the KTG motif of PBP5, known as the 'morphology maintenance domain ' (Ghosh & Young, 2003). Apart from maintaining cell shape, PBP5 also plays an important role in maintaining the intrinsic beta-lactam resistance in E. coli (Sarkar et al., 2010). The absence of PBP5 sensitizes the E. coli cell to beta-lactam antibiotics, while in trans expression of the same helps to reverse the lost resistance (Sarkar et al., 2010). The high copy number of PBP5 at the exponential phase of growth may be responsible for trapping the beta-lactam antibiotics, which prevents them from binding to the essential PBPs (...

High adaptability of the omega loop underlies the substrate-spectrum-extension evolution of a class A β-lactamase, PenL

“…From an evolutionary perspective, the omega loop of class A β-lactamases is a newly emerged structural element added to an ancestral penicillin binding protein (PBP), providing a deacylation activity embedded in the omega loop to evolve the PBP into a class A β-lactamase3738. The successful evolution of an enzyme may rely on the manipulation of the active site loops while the scaffold and catalytic activity of the ancestral enzyme are maintained39.…”

MSMEG_2432 of Mycobacterium smegmatis mc2155 is a dual function enzyme that exhibits DD-carboxypeptidase and β-lactamase activities

“…According to the 3D-model (see the legend of Fig. S3), the structure of the MSMEG_2432 has the common functional motifs of dd-CPases and β-lactamases, SXXK(tetrad), SXN(triad) and KTG (triad), which are supposedly responsible for the dual enzymatic activities as reported previously [10,19]. The polar networking of the residues present in the active-site helps in catalytic activity of these enzymes as evident in MSMEG_2432 (Fig.…”

“…S1). Apart from the active-site residues, PBPs carry an omegalike loop, covering the top portion of active-site groove, which is hypothesized to play a significant role during deacylation by recruiting the water molecule [19]. In silico 3D homology model of MSMEG_2432 was created using modeller v9.16 [31] (Fig.…”

“…An array of extensively studied dd-CPases (PBP4, 5, 6 and 6b) are reported in E. coli, though the reports on mycobacterial dd-CPases are limited. PBP5 of E. coli helps the cells to recover from morphological deformities in septuple PBP mutant E. coli where L182 in its omega loop plays an imperative role in its activity [16,19]. A previous study from our laboratory showed that the deletion of MSMEG_2433 and MSMEG_2432 changed the cell shape and length along with the nature of PG cross-link formation in M. smegmatis [15].…”

Glycosyltransferases and Transpeptidases/Penicillin-Binding Proteins: Valuable Targets for New Antibacterials

“…Generally, two HMW-PBPs (one class A and one class B) are necessary for the bacterium to survive, whereas deletion of all LMW-PBPs (class C), although deleterious, is not fatal to the bacterium [ 27 ]. As a result, HMW-PBPs are the primary targets of β-lactam antibiotics but LMW-PBPs may participate in the sensitization of the bacterium to these antibiotics [ 29 , 30 ] and simultaneous inhibition of HMW and LMW-PBPs may present an interesting synergistic effect.…”