Substitution of Alanine at Position 184 with Glutamic Acid in Escherichia coli PBP5 Ω-Like Loop Introduces a Moderate Cephalosporinase Activity.

Escherichia coli PBP5, a DD-carboxypeptidase (DD-CPase), helps in maintaining cell shape and intrinsic ß-lactam resistance. Though PBP5 does not have ß-lactamase activity under physiological pH, it has a common but shorter Ω-like loop resembling class A ß-lactamases. However, such Ω-like loop lacks the key glutamic acid residue that is present in ß-lactamases. It is speculated that ß-lactamases and DD-CPases might have undergone divergent evolution leading to distinct enzymes with different substrate specificities and functions indicating the versatility of the Ω-loops. Nonetheless, direct experimental evidence favoring the idea is insufficient. Here, aiming to investigate the effect of introducing a glutamic acid residue in the PBP5 Ω-like loop, we substituted A184 to E to create PBP5_A184E. Expression of PBP5_A184E in E. coli ∆PBP5 mutant elevates the ß-lactam resistance, especially for cephalosporins. However, like PBP5, PBP5_A184E has the ability to complement the aberrantly shaped E. coli septuple PBP mutant indicating an unaffected in vivo DD-CPase activity. Biochemical and bioinformatics analyses have substantiated the dual enzyme nature of the mutated enzyme possessing both DD-CPase and ß-lactamase activities. Therefore, substitution of A184 to E of Ω-like loop alone can introduce the cephalosporinase activity in E. coli PBP5 supporting the phenomenon of a single amino acid polymorphism.

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

DD-carboxypeptidases (DD-CPases) are low-molecular-mass (LMM) penicillin-binding proteins (PBPs) that are mainly involved in peptidoglycan remodelling, but little is known about the dd-CPases of mycobacteria. In this study, a putative DD-CPase of Mycobacterium smegmatis, MSMEG_2433 is characterized. The gene for the membrane-bound form of MSMEG_2433 was cloned and expressed in Escherichia coli in its active form, as revealed by its ability to bind to the Bocillin-FL (fluorescent penicillin). Interestingly, in vivo expression of MSMEG_2433 could restore the cell shape oddities of the septuple PBP mutant of E. coli, which was a prominent physiological characteristic of DD-CPases. Moreover, expression of MSMEG_2433 in trans elevated beta-lactam resistance in PBP deletion mutants (ΔdacAdacC) of E. coli, strengthening its physiology as a dd-CPase. To confirm the biochemical reason behind such physiological behaviours, a soluble form of MSMEG_2433 (sMSMEG_2433) was created, expressed and purified. In agreement with the observed physiological phenomena, sMSMEG_2433 exhibited DD-CPase activity against artificial and peptidoglycan-mimetic DD-CPase substrates. To our surprise, enzymic analyses of MSMEG_2433 revealed efficient deacylation for beta-lactam substrates at physiological pH, which is a unique characteristic of beta-lactamases. In addition to the MSMEG_2433 active site that favours dd-CPase activity, in silico analyses also predicted the presence of an omega-loop-like region in MSMEG_2433, which is an important determinant of its beta-lactamase activity. Based on the in vitro, in vivo and in silico studies, we conclude that MSMEG_2433 is a dual enzyme, possessing both DD-CPase and beta-lactamase activities.

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.

Penicillin-binding protein 5 (PBP5), a dd-carboxypeptidase, maintains cell shape and intrinsic beta-lactam resistance in E. coli. A strain lacking PBP5 loses intrinsic beta-lactam resistance and simultaneous lack of two other PBPs results in aberrantly shaped cells. PBP5 expression in trans complements the shape and restores the lost beta-lactam resistance. PBP5 has an 'Ω-loop'-like region similar to that in class A beta-lactamases. It was previously predicted that Leu182 present in the 'Ω-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.

PBP deletion mutants of Escherichia coli exhibit irregular distribution of MreB at the deformed zones.

MreB is a cytoskeletal protein, which is responsible for maintaining proper cellular morphology and is essential for cell survival. Likewise, penicillin-binding protein 5 (PBP5) helps in maintaining cell shape, though non-essential for survival. The contradicting feature of these two proteins paves the way for this study, wherein we attempt to draw a relation on the nature of distribution of MreB in PBP deletion mutants. The study revealed that the uniform MreB helices/patches were destabilized/disturbed at the zone of deformities of the PBP mutants, whereas the helical patterns were retained at the regions maintaining a rod shape. We interpret that MreB remains functional irrespective of its distribution being misguided by the aberrant shapes of PBP mutants.

The dipeptidyl peptidase IV inhibitors vildagliptin and K-579 inhibit a phospholipase C: a case of promiscuous scaffolds in proteins.

The long term side effects of any newly introduced drug is a subject of intense research, and often raging controversies. One such example is the dipeptidyl peptidase-IV (DPP4) inhibitor used for treating type 2 diabetes, which is inconclusively implicated in increased susceptibility to acute pancreatitis. Previously, based on a computational analysis of the spatial and electrostatic properties of active site residues, we have demonstrated that phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus is a prolyl peptidase using in vivo experiments. In the current work, we first report the inhibition of the native activity of PI-PLC by two DPP4 inhibitors - vildagliptin (LAF-237) and K-579. While vildagliptin inhibited PI-PLC at micromolar concentrations, K-579 was a potent inhibitor even at nanomolar concentrations. Subsequently, we queried a comprehensive, non-redundant set of 5000 human proteins (50% similarity cutoff) with known structures using serine protease (SPASE) motifs derived from trypsin and DPP4. A pancreatic lipase and a gastric lipase are among the proteins that are identified as proteins having promiscuous SPASE scaffolds that could interact with DPP4 inhibitors. The presence of such scaffolds in human lipases is expected since they share the same catalytic mechanism with PI-PLC. However our methodology also detects other proteins, often with a completely different enzymatic mechanism, that have significantly congruent domains with the SPASE motifs. The reported elevated levels of serum lipase, although contested, could be rationalized by inhibition of lipases reported here. In an effort to further our understanding of the spatial and electrostatic basis of DPP4 inhibitors, we have also done a comprehensive analysis of all 76 known DPP4 structures liganded to inhibitors till date. Also, the methodology presented here can be easily adopted for other drugs, and provide the first line of filtering in the identification of pathways that might be inadvertently affected due to promiscuous scaffolds in proteins.

Sub-inhibitory cefsulodin sensitization of E. coli to ß-lactams is mediated by PBP1b inhibition.

The combination of antibiotics is one of the strategies to combat drug-resistant bacteria, though only a handful of such combinations are in use, such as the ß-lactam combinations. In the present study, the efficacy of a specific sub-inhibitory concentration of cefsulodin with other ß-lactams was evaluated against a range of Gram-negative clinical isolates. This approach increased the sensitivity of the isolates, regardless of the ß-lactamase production. The preferred target and mechanism of action of cefsulodin were identified in laboratory strains of Escherichia coli, by examining the effects of deleting the penicillin-binding protein (PBP) 1a and 1b encoding genes individually. Deletion of PBP1b was involved in sensitizing the bacteria to ß-lactam agents, irrespective of its O-antigen status. Moreover, the use of a sub-inhibitory concentration of cefsulodin in combination with a ß-lactam exerted an effect similar to that one obtained for PBP1b gene deletion. We conclude that the identified ß-lactam/cefsulodin combination works by inhibiting PBP1b (at least partially) despite the involvement of ß-lactamases, and therefore could be extended to a broad range of Gram-negative pathogens.

Moderate deacylation efficiency of DacD explains its ability to partially restore beta-lactam resistance in Escherichia coli PBP5 mutant.

Of the five dd-carboxypeptidases in Escherichia coli, only PBP5 demonstrates its physiological significance by maintaining cell shape and intrinsic beta-lactam resistance. DacD can partially compensate for the lost beta-lactam resistance in PBP5 mutant, although its biochemical reason is unclear. To understand the mechanism(s) underlying such behaviour, we constructed soluble DacD (sDacD) and compared its biophysical and biochemical properties with those of sPBP5, in vitro. Unlike sPBP6, sDacD can deacylate Bocillin significantly, which is very similar to sPBP5. sDacD shows weak dd-carboxypeptidase activity, although lower than that of sPBP5. Bioinformatics analyses reveal a similar architecture of sPBP5 and sDacD. Therefore, based on the obtained results we can infer that biochemically DacD and PBP5 are more closely related to each other than to PBP6, enabling DacD and PBP5 to play a nearly similar physiological function in terms of recovering the lost beta-lactam resistance.

PBP5, PBP6 and DacD play different roles in intrinsic ß-lactam resistance of Escherichia coli.

Escherichia coli PBP5, PBP6 and DacD, encoded by dacA, dacC and dacD, respectively, share substantial amino acid identity and together constitute ~50 % of the total penicillin-binding proteins of E. coli. PBP5 helps maintain intrinsic ß-lactam resistance within the cell. To test if PBP6 and DacD play simlar roles, we deleted dacC and dacD individually, and dacC in combination with dacA, from E. coli 2443 and compared ß-lactam sensitivity of the mutants and the parent strain. ß-Lactam resistance was complemented by wild-type, but not dd-carboxypeptidase-deficient PBP5, confirming that enzymic activity of PBP5 is essential for ß-lactam resistance. Deletion of dacC and expression of PBP6 during exponential or stationary phase did not alter ß-lactam resistance of a dacA mutant. Expression of DacD during mid-exponential phase partially restored ß-lactam resistance of the dacA mutant. Therefore, PBP5 dd-carboxypeptidase activity is essential for intrinsic ß-lactam resistance of E. coli and DacD can partially compensate for PBP5 in this capacity, whereas PBP6 cannot.