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

Chowdhury, Chiranjit; Kar, Debasish; Dutta, Mouparna; Kumar, Amresh; Ghosh, Anindya S.

doi:10.1111/1574-6968.12009

Cited by 14 publications

(28 citation statements)

References 40 publications

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“…DacD is a periplasmic LMM PBP endowed with low DD-carboxypeptidase activity that is ubiquitously distributed among Gram-negative bacterial species, but a well-defined function has still not be identified (15)(16)(17)(18)(19)(20). LMM PBPs displaying DD-carboxypeptidase activity are generally thought to be modulators of bacterial morphology and, in fact, mutational losses of the LMM PBP5 (DacA) in E. coli can result in morphological alterations (15)(16)(17)(18)(19)(20).…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…LMM PBPs displaying DD-carboxypeptidase activity are generally thought to be modulators of bacterial morphology and, in fact, mutational losses of the LMM PBP5 (DacA) in E. coli can result in morphological alterations (15)(16)(17)(18)(19)(20). DacD displays around 48% sequence identity with DacA, but it contains larger amounts of ␤-sheets and lower DD-carboxypeptidase activity (15)(16)(17)(18)(19)(20). Moreover, unlike loss of DacA, mutational loss of E. coli DacD resulted in no apparent phenotypic consequences, thus suggesting different physiological roles for these LMM PBPs (15)(16)(17)(18)(19)(20).…”

Section: Discussionmentioning

confidence: 99%

“…DacD displays around 48% sequence identity with DacA, but it contains larger amounts of ␤-sheets and lower DD-carboxypeptidase activity (15)(16)(17)(18)(19)(20). Moreover, unlike loss of DacA, mutational loss of E. coli DacD resulted in no apparent phenotypic consequences, thus suggesting different physiological roles for these LMM PBPs (15)(16)(17)(18)(19)(20). In this context, different experimental results suggest roles for the LMM PBP DD-carboxypeptidases in processes other than morphology maintenance such as manipulations of PG structure to modulate biofilm growth and even host immune responses (15)(16)(17)(18)(19)(20).…”

Section: Discussionmentioning

confidence: 99%

“…This phenotype could be associated with the deletion of the dacD gene in S. enterica and also in E. coli, concomitant with a lower accumulation of GOB-18 in the periplasm of the ⌬dacD mutants. dacD encodes the low-molecular-mass (LMM) penicillin binding protein 6b (PBP6b), ubiquitously present in Gram-negative bacteria but of still poorly defined function(s) (15)(16)(17)(18)(19)(20). Moreover, the ⌬dacD mutants of both species showed altered biofilm growth.…”

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Low-Molecular-Mass Penicillin Binding Protein 6b (DacD) Is Required for Efficient GOB-18 Metallo-β-Lactamase Biogenesis in Salmonella enterica and Escherichia coli

Brambilla

Morán-Barrio

Viale

2014

Antimicrob Agents Chemother

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Metallo-␤-lactamases (MBLs) are Zn 2؉-containing secretory enzymes of clinical relevance, whose final folding and metal ion assembly steps in Gram-negative bacteria occur after secretion of the apo form to the periplasmic space. In the search of periplasmic factors assisting MBL biogenesis, we found that dacD null (⌬dacD) mutants of Salmonella enterica and Escherichia coli expressing the pre-GOB-18 MBL gene from plasmids showed significantly reduced resistance to cefotaxime and concomitant lower accumulation of GOB-18 in the periplasm. This reduced accumulation of GOB-18 resulted from increased accessibility to proteolytic attack in the periplasm, suggesting that the lack of DacD negatively affects the stability of secreted apo MBL forms. Moreover, ⌬dacD mutants of S. enterica and E. coli showed an altered ability to develop biofilm growth. DacD is a widely distributed low-molecular-mass (LMM) penicillin binding protein (PBP6b) endowed with low DD-carboxypeptidase activity whose functions are still obscure. Our results indicate roles for DacD in assisting biogenesis of particular secretory macromolecules in Gram-negative bacteria and represent to our knowledge the first reported phenotypes for bacterial mutants lacking this LMM PBP.␤-Lactamase production represents a common mechanism of bacterial resistance and cause of failure in the treatment of infections (1). Among the different groups of ␤-lactamases defined on the basis of structural similarity and catalytic mechanisms, the metallo-␤-lactamases (MBLs) are especially worrisome (2, 3). This group is constituted entirely of metalloenzymes employing Zn 2ϩ for catalysis; members of this group can hydrolyze a broad spectrum of substrates, including the latest generations of clinically relevant ␤-lactams, and can be rapidly spread by horizontal gene transfer (1-3). Among the different strategies for controlling MBL dissemination, efforts have so far been focused on the design of a general MBL inhibitor, a goal so far hindered by the diversity of active-site structures among these metalloenzymes (4).A less known aspect of MBL research, but one that may provide potential targets for antimicrobial drug design, is the MBL biogenesis pathway (5). We previously reported that the productive biogenesis of the GOB-18 MBL in Escherichia coli requires an "expanded" DnaK chaperone system to assist the cytoplasmic transit of the preapoprotein to the secretion system (5). Moreover, we also found that secretion of the apo GOB-18 form is driven by the Sec (SecA-SecYEG) machinery, implying that final folding and Zn 2ϩ ion assembly to the native MBL conformation occur in the periplasm (5). Unfolded proteins emerging from the Sec channel are greeted by a highly complex macromolecule-crowded environment containing an array of modifying enzymes, some of them endowed with the ability to act as folding assistants (6-8). This complex scenario can certainly affect individual folding landscapes (6), but whether the final steps of MBL folding and Zn 2ϩ assembly are assisted by periplasmic ...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Low-Molecular-Mass Penicillin Binding Protein 6b (DacD) Is Required for Efficient GOB-18 Metallo-β-Lactamase Biogenesis in Salmonella enterica and Escherichia coli

Brambilla

Morán-Barrio

Viale

2014

Antimicrob Agents Chemother

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A Tyrosine Residue Along with a Glutamic Acid of the Omega-Like Loop Governs the Beta-Lactamase Activity of MSMEG_4455 in Mycobacterium smegmatis

et al. 2017

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Mycobacterial beta-lactamases are involved in exerting beta-lactam resistance, though many of these proteins remain uncharacterized. Here, we have characterized MSMEG_4455 of Mycobacterium smegmatis as a beta-lactamase using molecular, biochemical and mutational techniques. To elucidate its nature in vivo and in vitro, and to predict its structure-function relationship in silico analysis is done. The MSMEG_4455 is cloned and expressed ectopically in a beta-lactamase deficient Escherichia coli mutant to establish the in vivo beta-lactamase like nature via minimum inhibitory concentration (MIC) determination. Likewise the in vivo results, purified soluble form of MSMEG_4455 showed beta-lactam hydrolysis pattern similar to group 2a penicillinase. In silico analyses of MSMEG_4455 reveal glutamic acid (E)193 and tyrosine (Y)194 of omega-like loop might have importance in strengthening hydrogen bond network around the active-site, though involvement of tyrosine is rare for beta-lactamase activity. Accordingly, these residues are mutated to alanine (A) and phenylalanine (F), respectively. The mutated proteins have partially lost their ability to exert beta-lactamase activity both in vivo and in vitro. The Y194F mutation had more prominent effect on the enzymatic activity. Therefore, we infer that Y194 is the key for beta-lactamase activity of MSMEG_4455.

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

et al. 2018

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Escherichia coli PBP5, a DD-carboxypeptidase (DD-CPase), helps in maintaining cell shape and intrinsic β-lactam resistance. Though PBP5 does not have β-lactamase activity under physiological pH, it has a common but shorter Ω-like loop resembling class A β-lactamases. However, such Ω-like loop lacks the key glutamic acid residue that is present in β-lactamases. It is speculated that β-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 β-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 β-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.

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Moderate deacylation efficiency of DacD explains its ability to partially restore beta-lactam resistance inEscherichia coliPBP5 mutant

Cited by 14 publications

References 40 publications

Low-Molecular-Mass Penicillin Binding Protein 6b (DacD) Is Required for Efficient GOB-18 Metallo-β-Lactamase Biogenesis in Salmonella enterica and Escherichia coli

Low-Molecular-Mass Penicillin Binding Protein 6b (DacD) Is Required for Efficient GOB-18 Metallo-β-Lactamase Biogenesis in Salmonella enterica and Escherichia coli

A Tyrosine Residue Along with a Glutamic Acid of the Omega-Like Loop Governs the Beta-Lactamase Activity of MSMEG_4455 in Mycobacterium smegmatis

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

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