The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step

Kokkonen, Piia; Slanska, Michaela; Dockalova, Veronika; Pinto, Gaspar; Sánchez‐Carnerero, Esther M.; Damborský, Jiřı́; Klán, Petr; Prokop, Zbyněk; Bednář, David

doi:10.1016/j.csbj.2020.03.017

Cited by 16 publications

(18 citation statements)

References 36 publications

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“…In Staphylococcus aureus , PBPs form a stable covalent bond between their catalytic Ser370 residues and AMX, thereby preventing bacterial cell wall synthesis by inactivating the transpeptidase domain [ 33 ]. It is known that modification of amino acid residues lining the drug access tunnels affects the enzyme’s activity, specificity, enantioselectivity, and stability [ 34 , 35 ]. In case of enzymes, such as xylanase, with buried binding sites, transporting substrates between active sites and the surrounding solution, through the access tunnels is a critical step in the catalytic cycle of these enzymes.…”

Section: Discussionmentioning

confidence: 99%

The penicillin binding protein 1A of Helicobacter pylori, its amoxicillin binding site and access routes

et al. 2021

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Background Amoxicillin-resistant H. pylori strains are increasing worldwide. To explore the potential resistance mechanisms involved, the 3D structure modeling and access tunnel prediction for penicillin-binding proteins (PBP1A) was performed, based on the Streptococcus pneumoniae, PBP 3D structure. Molecular covalent docking was used to determine the interactions between amoxicillin (AMX) and PBP1A. Results The AMX-Ser368 covalent complex interacts with the binding site residues (Gly367, Ala369, ILE370, Lys371, Tyr416, Ser433, Thr541, Thr556, Gly557, Thr558, and Asn560) of PBP1A, non-covalently. Six tunnel-like structures, accessing the PBP1A binding site, were characterized, using the CAVER algorithm. Tunnel-1 was the ultimate access route, leading to the drug catalytic binding residue (Ser368). This tunnel comprises of eighteen amino acid residues, 8 of which are shared with the drug binding site. Subsequently, to screen the presence of PBP1A mutations, in the binding site and tunnel residues, in our clinical strains, in vitro assays were performed. H. pylori strains, isolated under gastroscopy, underwent AMX susceptibility testing by E-test. Of the 100 clinical strains tested, 4 were AMX-resistant. The transpeptidase domain of the pbp1a gene of these resistant, plus 10 randomly selected AMX-susceptible strains, were amplified and sequenced. Of the amino acids lining the tunnel-1 and binding site residues, three (Ser414Arg, Val469Met and Thr556Ser) substitutions, were detected in 2 of the 4 resistant and none of the sequenced susceptible strains, respectively. Conclusions We hypothesize that mutations in amino acid residues lining the binding site and/or tunnel-1, resulting in conformational/spatial changes, may block drug binding to PBP1A and cause AMX resistance.

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Section: Discussionmentioning

confidence: 99%

The penicillin binding protein 1A of Helicobacter pylori, its amoxicillin binding site and access routes

et al. 2021

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“…Previous studies indicated that the characteristics of substrate access tunnels can have a decisive influence on enzyme-substrate specificity and activity [53][54][55][56]. In t-PlaF, we focused on T1 and T2 because only these two allow direct access of GPL or LGPL substrates from the membrane in the t-PlaF A configuration.…”

Section: Discussionmentioning

confidence: 99%

“…The Trp substitutions decreased PlaF's lipolytic activity for small and large substrates only when introduced in T2, which suggests that T2 is involved in substrate access. However, from such steady-state experiments, it cannot be excluded that the Trp substitutions influence product egress, too [54].…”

Section: Discussionmentioning

confidence: 99%

Substrate access mechanism in a novel membrane-bound phospholipase A of Pseudomonas aeruginosa concordant with specificity and regioselectivity

Ahmad

Strunk

Schott‐Verdugo

et al. 2021

Preprint

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PlaF is a cytoplasmic membrane-bound phospholipase A1 from Pseudomonas aeruginosa that alters the membrane glycerophospholipid (GPL) composition and fosters the virulence of this human pathogen. PlaF activity is regulated by a dimer-to-monomer transition followed by tilting of the monomer in the membrane. However, how substrates reach the active site and how the characteristics of the active site tunnels determine the activity, specificity, and regioselectivity of PlaF for natural GPL substrates has remained elusive. Here, we combined unbiased and biased all-atom molecular dynamics (MD) simulations and configurational free energy computations to identify access pathways of GPL substrates to the catalytic center of PlaF. Our results map out a distinct tunnel through which substrates access the catalytic center. PlaF variants with bulky tryptophan residues in this tunnel revealed decreased catalysis rates due to tunnel blockage. The MD simulations suggest that GPLs preferably enter the active site with the sn-1 acyl chain first, which agrees with the experimentally demonstrated PLA1 activity of PlaF. We propose that the acyl chain-length specificity of PlaF is determined by the structural features of the access tunnel, which results in favorable free energy of binding of medium-chain GPLs. The suggested egress route conveys fatty acid products to the dimerization interface and, thus, contributes to understanding the product feedback regulation of PlaF by fatty acid-triggered dimerization. These findings open up opportunities for developing potential PlaF inhibitors, which may act as antibiotics against P. aeruginosa.

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“…The mutations of Ser284-Thr285-Ile286 and Phe291 to Ala lead to increased enzyme activity without an apparent effect on its structure and dimerization. It was concluded that these critical residues form a nano-channel acting as an allosteric regulator [ 219 , 220 , 221 ].…”

Section: Rna Virusesmentioning

confidence: 99%

Precursors of Viral Proteases as Distinct Drug Targets

Majerová

Novotny

2021

Viruses

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Viral proteases are indispensable for successful virion maturation, thus making them a prominent drug target. Their enzyme activity is tightly spatiotemporally regulated by expression in the precursor form with little or no activity, followed by activation via autoprocessing. These cleavage events are frequently triggered upon transportation to a specific compartment inside the host cell. Typically, precursor oligomerization or the presence of a co-factor is needed for activation. A detailed understanding of these mechanisms will allow ligands with non-canonical mechanisms of action to be designed, which would specifically modulate the initial irreversible steps of viral protease autoactivation. Binding sites exclusive to the precursor, including binding sites beyond the protease domain, can be exploited. Both inhibition and up-regulation of the proteolytic activity of viral proteases can be detrimental for the virus. All these possibilities are discussed using examples of medically relevant viruses including herpesviruses, adenoviruses, retroviruses, picornaviruses, caliciviruses, togaviruses, flaviviruses, and coronaviruses.

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The impact of tunnel mutations on enzymatic catalysis depends on the tunnel-substrate complementarity and the rate-limiting step

Cited by 16 publications

References 36 publications

The penicillin binding protein 1A of Helicobacter pylori, its amoxicillin binding site and access routes

The penicillin binding protein 1A of Helicobacter pylori, its amoxicillin binding site and access routes

Substrate access mechanism in a novel membrane-bound phospholipase A of Pseudomonas aeruginosa concordant with specificity and regioselectivity

Precursors of Viral Proteases as Distinct Drug Targets

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