Effect of mutation at oxyanion hole residu (H110F) on activity of Lk4 lipase

Abstract Rhomboids are ubiquitous intramembrane serine proteases involved in various signaling pathways. While the high-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed an active site comprised of a serine-histidine dyad and an extensive oxyanion hole, the molecular details of rhomboid catalysis were unclear because substrates are unknown for most of the family members. Here we used the only known physiological pair of AarA rhomboid with its psTatA substrate to decipher the contribution of catalytically important residues to the reaction rate enhancement. An MD-refined homology model of AarA was used to identify residues important for catalysis. We demonstrated that the AarA active site geometry is strict and intolerant to alterations. We probed the roles of H83 and N87 oxyanion hole residues and determined that substitution of H83 either abolished AarA activity or reduced the transition state stabilization energy (ΔΔG‡) by 3.1 kcal/mol; substitution of N87 decreased ΔΔG‡ by 1.6–3.9 kcal/mol. Substitution M154, a residue conserved in most rhomboids that stabilizes the catalytic general base, to tyrosine, provided insight into the mechanism of nucleophile generation for the catalytic dyad. This study provides a quantitative evaluation of the role of several residues important for hydrolytic efficiency and oxyanion stabilization during intramembrane proteolysis.

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13C-NMR study of the inhibition of δ-chymotrypsin by a tripeptide-glyoxal inhibitor

Biochemical Journal ◽

10.1042/bj3620339 ◽

2002 ◽

Vol 362 (2) ◽

pp. 339-347 ◽

Cited By ~ 7

Author(s):

Aleksandra DJURDJEVIC-PAHL ◽

Chandralal HEWAGE ◽

J. Paul G. MALTHOUSE

Keyword(s):

13C Nmr ◽

Exchange Process ◽

Competitive Inhibitor ◽

Minor Role ◽

Oxyanion Hole ◽

Inhibitor Complex ◽

Rapid Exchange ◽

Nmr Study ◽

A Minor ◽

Hydroxy Groups

A new inhibitor, Z-Ala-Pro-Phe-glyoxal (where Z is benzyloxycarbonyl),has been synthesized and shown to be a competitive inhibitor of δ-chymotrypsin, with a Ki of 25±8nM at pH7.0 and 25°C. Z-Ala-Pro-[1-13C]Phe-glyoxal and Z-Ala-Pro-[2-13C]Phe-glyoxal have been synthesized, and 13C-NMR has been used to determine how they interact with δ-chymotrypsin. Using Z-Ala-Pro-[2-13C]Phe-glyoxal we have detected a signal at 100.7p.p.m. which we assign to the tetrahedral adduct formed between the hydroxy group of Ser-195 and the 13C-enriched keto-carbon of the inhibitor. This signal is in a pH-dependent slow exchange with a signal at 107.6p.p.m. which depends on a pKa of ∼ 4.5, which we assign to oxyanion formation. Thus we are the first to detect an oxyanion pKa in a reversible chymotrypsin—inhibitor complex. A smaller titration shift of 100.7p.p.m. to 103.9p.p.m. with a pKa of ∼ 5.3 is also detected due to a rapid exchange process. This pKa is also detected with the Z-Ala-Pro-[1-13C]Phe-glyoxal inhibitor and gives a larger titration shift of 91.4p.p.m. to 97.3p.p.m., which we assign to the ionization of the hydrated aldehyde hydroxy groups of the enzyme-bound inhibitor. Protonation of the oxyanion in the oxyanion hole decreases the binding efficiency of the inhibitor. From this decrease in binding efficiency we estimate that oxyanion binding in the oxyanion hole reduces the oxyanion pKa by 1.3 pKa units. We calculate that the pKas of the oxyanions of the hemiketal and hydrated aldehyde moieties of the glyoxal inhibitor are both lowered by 6.4–6.9 pKa units on binding to chymotrypsin. Therefore we conclude that oxyanion binding in the oxyanion hole has only a minor role in decreasing the oxyanion pKa. We also investigate how the inhibitor breaks down at alkaline pH, and how it breaks down at neutral pH in the presence of chymotrypsin.

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MD simulations reveal alternate conformations of the oxyanion hole in the Zika virus NS2B/NS3 protease

Proteins Structure Function and Bioinformatics ◽

10.1002/prot.25809 ◽

2019 ◽

Vol 88 (2) ◽

pp. 345-354

Author(s):

Jinhong Ren ◽

Hyun Lee ◽

Alpa Kotak ◽

Michael E. Johnson

Keyword(s):

Zika Virus ◽

Md Simulations ◽

Oxyanion Hole ◽

Ns3 Protease

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Substitutions at Position 105 in SHV Family β-Lactamases Decrease Catalytic Efficiency and Cause Inhibitor Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00711-12 ◽

2012 ◽

Vol 56 (11) ◽

pp. 5678-5686 ◽

Cited By ~ 6

Author(s):

Mei Li ◽

Benjamin C. Conklin ◽

Magdalena A. Taracila ◽

Rebecca A. Hutton ◽

Marion J. Skalweit

Keyword(s):

Clavulanic Acid ◽

Susceptibility Testing ◽

Catalytic Efficiency ◽

Wild Type ◽

Oxyanion Hole ◽

Content Type ◽

Class A ◽

Inhibitor Resistance ◽

Structural Probes ◽

Variant Residue

ABSTRACTAmbler position 105 in class A β-lactamases is implicated in resistance to clavulanic acid, although no clinical isolates with mutations at this site have been reported. We hypothesized that Y105 is important in resistance to clavulanic acid because changes in positioning of the inhibitor for ring oxygen protonation could occur. In addition, resistance to bicyclic 6-methylidene penems, which are interesting structural probes that inhibit all classes of serine β-lactamases with nanomolar affinity, might emerge with substitutions at position 105, especially with nonaromatic substitutions. All 19 variants of SHV-1 with variations at position 105 were prepared. Antimicrobial susceptibility testing showed thatEscherichia coliDH10B expressing Y105 variants retained activity against ampicillin, except for the Y105L variant, which was susceptible to all β-lactams, similar to the case for the host control strain. Several variants had elevated MICs to ampicillin-clavulanate. However, all the variants remained susceptible to piperacillin in combination with a penem inhibitor (MIC, ≤2/4 mg/liter). The Y105E, -F, -M, and -R variants demonstrated reduced catalytic efficiency toward ampicillin compared to the wild-type (WT) enzyme, which was caused by increasedKm. Clavulanic acid and penemKivalues were also increased for some of the variants, especially Y105E. Mutagenesis at position 105 in SHV yields mutants resistant to clavulanate with reduced catalytic efficiency for ampicillin and nitrocefin, similar to the case for the class A carbapenemase KPC-2. Our modeling analyses suggest that resistance is due to oxyanion hole distortion. Susceptibility to a penem inhibitor is retained although affinity is decreased, especially for the Y105E variant. Residue 105 is important to consider when designing new inhibitors.

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Crystal Structures of KPC-2 β-Lactamase in Complex with 3-Nitrophenyl Boronic Acid and the Penam Sulfone PSR-3-226

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.06099-11 ◽

2012 ◽

Vol 56 (5) ◽

pp. 2713-2718 ◽

Cited By ~ 23

Author(s):

Wei Ke ◽

Christopher R. Bethel ◽

Krisztina M. Papp-Wallace ◽

Sundar Ram Reddy Pagadala ◽

Micheal Nottingham ◽

...

Keyword(s):

Crystal Structures ◽

Active Site ◽

Boronic Acid ◽

Carbonyl Oxygen ◽

Oxyanion Hole ◽

Covalent Interaction ◽

Class A ◽

Inactivation Rate Constant ◽

Reversible Covalent ◽

Acid Compound

ABSTRACTClass A carbapenemases are a major threat to the potency of carbapenem antibiotics. A widespread carbapenemase, KPC-2, is not easily inhibited by β-lactamase inhibitors (i.e., clavulanic acid, sulbactam, and tazobactam). To explore different mechanisms of inhibition of KPC-2, we determined the crystal structures of KPC-2 with two β-lactamase inhibitors that follow different inactivation pathways and kinetics. The first complex is that of a small boronic acid compound, 3-nitrophenyl boronic acid (3-NPBA), bound to KPC-2 with 1.62-Å resolution. 3-NPBA demonstrated aKmvalue of 1.0 ± 0.1 μM (mean ± standard error) for KPC-2 and blocks the active site by making a reversible covalent interaction with the catalytic S70 residue. The two boron hydroxyl atoms of 3-NPBA are positioned in the oxyanion hole and the deacylation water pocket, respectively. In addition, the aromatic ring of 3-NPBA provides an edge-to-face interaction with W105 in the active site. The structure of KPC-2 with the penam sulfone PSR-3-226 was determined at 1.26-Å resolution. PSR-3-226 displayed aKmvalue of 3.8 ± 0.4 μM for KPC-2, and the inactivation rate constant (kinact) was 0.034 ± 0.003 s−1. When covalently bound to S70, PSR-3-226 forms atrans-enamine intermediate in the KPC-2 active site. The predominant active site interactions are generated via the carbonyl oxygen, which resides in the oxyanion hole, and the carboxyl moiety of PSR-3-226, which interacts with N132, N170, and E166. 3-NPBA and PSR-3-226 are the first β-lactamase inhibitors to be trapped as an acyl-enzyme complex with KPC-2. The structural and inhibitory insights gained here could aid in the design of potent KPC-2 inhibitors.

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