Acyl-enzyme dynamics, tautomerisation and hydration regulate turnover of carbapenem antibiotics by the OXA-48 β-lactamase

This study combines X-ray crystallography and molecular dynamics simulations to reveal that the turnover of carbapenem antibiotics by OXA-48 $\beta$-lactamase is regulated by a complex interplay of acyl-enzyme tautomerisation, conformational dynamics, and hydration, with the OXA-519 variant showing enhanced hydrolytic potential due to a single active-site substitution.

The Gist

The Big Picture: A Microscopic Lock and Key Battle

Imagine your body is a fortress, and bacteria are the invaders trying to break in. To stop them, doctors use powerful weapons called antibiotics. One of the strongest types of antibiotics is the carbapenem family (like Meropenem and Ertapenem). These are the "special forces" used when other antibiotics fail.

However, some bacteria have evolved a shield. They produce a tiny machine called an enzyme named OXA-48. Think of OXA-48 as a security guard inside the bacterial cell. Its job is to spot the antibiotic "special forces," grab them, and cut them in half so they can't hurt the bacteria. This is how bacteria become resistant to medicine.

This paper is a deep dive into exactly how this security guard (OXA-48) catches and destroys the antibiotic. The scientists also looked at a "super-guard" version called OXA-519, which is even better at destroying these specific drugs.

---

The Mechanism: The "Velcro Trap"

To understand the study, imagine the antibiotic as a piece of Velcro with a sticky side.

1. The Trap (Acylation): When the antibiotic enters the bacteria, the OXA-48 guard grabs it. The antibiotic sticks to the guard's hand (a specific amino acid called Serine). Now, the antibiotic is stuck in a "Velcro trap." It's not dead yet, but it's stuck.
2. The Shape-Shifter (Tautomerization): This is the tricky part. Once stuck, the antibiotic can twist and change its shape slightly, like a gymnast doing a flip. It can land in two main poses:
   • The "Delta-2" Pose: A shape that looks ready to be finished off.
   • The "Delta-1" Pose: A shape that is a bit awkward and harder to finish.
3. The Finish (Deacylation): To kill the antibiotic, the guard needs to bring in a water molecule (like a tiny hammer) to smash the bond holding the antibiotic to the hand. If the water hits the right spot at the right angle, the antibiotic breaks free and is destroyed.

What the Scientists Found

The researchers used two main tools: X-ray Crystallography (taking super-sharp 3D photos of the guard holding the antibiotic) and Molecular Dynamics Simulations (a high-speed movie of the guard and antibiotic moving around in a computer).

Here are the key discoveries:

1. The "Gymnast" Needs the Right Pose

The study found that the antibiotic doesn't just sit still. It wiggles and changes shape.

• The Finding: The "Delta-2" shape is the one that makes it easiest for the water hammer to hit the antibiotic. The "Delta-1" shapes are like the gymnast doing a backflip in the wrong direction; they make it hard for the water to hit the target.
• The Analogy: Imagine trying to hit a moving target with a dart. If the target is spinning the right way (Delta-2), you can hit it. If it's spinning the wrong way (Delta-1), you miss. The OXA-48 guard is actually quite good at waiting for the antibiotic to spin into the "Delta-2" position before it strikes.

2. The "Water Channel" Problem

For the guard to smash the antibiotic, a specific water molecule needs to slide into a tiny tunnel (the "deacylating water channel") to reach the antibiotic.

• The Problem: In the standard OXA-48 guard, there is a small gatekeeper (a piece of the protein called Valine-120) that often blocks this tunnel. It's like a bouncer standing in the doorway, preventing the water hammer from getting close enough to do its job.
• The Result: Because the water has trouble getting in, the antibiotic sometimes stays stuck to the guard for too long, or the guard gets tired and stops working.

3. The "Super-Guard" (OXA-519)

The scientists also studied a mutant version of the guard, OXA-519. This version has a tiny change: the bouncer (Valine) was swapped for a slightly different person (Leucine).

• The Change: This new bouncer (Leucine) stands in a different position. It doesn't block the doorway as much.
• The Result: The water hammer can now slide in much more easily. The OXA-519 guard is much faster and more efficient at destroying the antibiotic. It's like the bouncer stepped aside, letting the water hammer strike the antibiotic instantly.

4. The "Beta-Lactone" Detour

Sometimes, instead of using the water hammer, the antibiotic tries to fix itself by twisting its own arm (the 6α-hydroxyethyl group) to break the bond. This creates a different shape called a beta-lactone.

• The Finding: The OXA-519 guard is surprisingly good at letting the antibiotic do this self-fixing twist. In fact, the OXA-519 guard is so efficient that it often uses this "self-fix" method to get rid of the antibiotic faster than the standard guard.
• The Twist: Once the antibiotic twists into this beta-lactone shape, it gets stuck in the guard's hand again, but this time it's harder to get out. It's like the antibiotic got tangled in its own shoelaces.

Why Does This Matter?

This research is like a blueprint for designing better weapons.

• Understanding the Enemy: By knowing exactly how the guard catches the antibiotic (the shapes it takes, where the water goes), scientists can design new antibiotics that the guard can't catch.
• The "Lock" Strategy: If we can design an antibiotic that forces the guard into the "Delta-1" shape (the awkward pose) or blocks the water tunnel permanently, the guard will fail, and the bacteria will die.
• The Future: The study suggests that if we can make antibiotics that restrict their own movement (so they can't twist into the "good" shape for the guard) or that fit perfectly into the "water channel" to jam it, we can overcome this resistance.

Summary in One Sentence

This paper reveals that the bacterial enzyme OXA-48 destroys antibiotics by waiting for them to twist into a specific shape and then using a water molecule to break them apart, but a mutated version (OXA-519) is even faster because it keeps the "water door" open, giving scientists new clues on how to design drugs that jam this mechanism.

Technical Summary

1. Problem Statement

The OXA-48 class D serine β-lactamase (SBL) is a globally disseminated enzyme responsible for resistance to carbapenems, the most potent antibiotics against Enterobacterales. While OXA-48 hydrolyzes many β-lactams, it turns over 1β-methyl carbapenems (e.g., meropenem, ertapenem) relatively slowly. The molecular mechanisms governing the deacylation step (the rate-limiting step) of these specific substrates remain poorly understood. Specifically, the roles of:

• Acyl-enzyme tautomerisation: The interconversion between $\Delta^2$-enamine and $\Delta^1$-imine forms of the carbapenem-derived intermediate.
• Conformational dynamics: The orientation of the 6$\alpha$-hydroxyethyl group.
• Active site hydration: The positioning of water molecules relative to the catalytic base (carboxylated Lys73, KCX73).
• Alternative pathways: The competition between hydrolytic deacylation and reversible $\beta$-lactone formation.

Furthermore, the OXA-519 variant (a natural OXA-48 mutant with a Val120Leu substitution) exhibits significantly enhanced turnover rates for these carbapenems, yet the structural basis for this hyper-activity is unclear.

2. Methodology

The authors employed a multi-scale structural biology and computational approach:

• X-ray Crystallography:

  • Determined high-resolution structures (1.29–1.95 Å) of OXA-48 and OXA-519 in complex with meropenem and ertapenem.
  • Utilized time-series soaking experiments (1 hour, 2 hours, 4 hours, 16–22 hours) to capture transient intermediates (acyl-enzymes) and final products (hydrolyzed or $\beta$-lactone).
  • Analyzed tautomeric states ($\Delta^1$-imine vs. $\Delta^2$-enamine) and the protonation/carboxylation status of the catalytic Lys73.

• Molecular Dynamics (MD) Simulations:

  • Conducted extensive MD simulations (totaling 1.5 $\mu$s) of OXA-48 and OXA-519 acyl-enzyme complexes in all three tautomeric states ($\Delta^2$, $\Delta^1(S)$, $\Delta^1(R)$).
  • Analyzed root-mean-square fluctuation (RMSF), hydrogen bonding networks, and the positioning of the deacylating water (DW).
  • Calculated the probability of the 6$\alpha$-hydroxyethyl group adopting specific conformations (I, II, III) and the Bürgi-Dunitz trajectory angles for nucleophilic attack.

• QM/MM Simulations:

  • Used Quantum Mechanics/Molecular Mechanics (QM/MM) to model the formation of hydrolyzed and $\beta$-lactone products from the acyl-enzyme intermediate.
  • Ran subsequent MD simulations on these product complexes to assess their retention and mobility within the active site.

3. Key Contributions and Results

A. Structural Characterization of OXA-48 Acyl-Enzymes

• Tautomer Diversity: The study identified acyl-enzymes in both $\Delta^1$-imine and $\Delta^2$-enamine forms. Notably, it captured the $\Delta^1(R)$ configuration of ertapenem for the first time.
• 6$\alpha$-Hydroxyethyl Conformations: Crystal structures revealed the 6$\alpha$-hydroxyethyl group in Conformation III (~290° dihedral angle), a state previously unobserved in OXA-48 acyl-enzymes (excluding inactive "flipped" intermediates). This conformation correlates with a carboxylated Lys73 and specific rotamers of Val120.
• Lys73 Status: Longer soaking times led to the decarboxylation of Lys73, suggesting a link between the catalytic cycle and the stability of the carboxylated state.

B. Dynamics and Deacylation Competence in OXA-48

• Tautomer Preference: MD simulations demonstrated that the $\Delta^2$-enamine tautomer is the most stable and least mobile form. Crucially, it adopts conformations most favorable for hydrolysis.
• Water Positioning: The $\Delta^2$-enamine form facilitates the positioning of the deacylating water molecule within 3 Å of the C7 carbonyl carbon (the nucleophilic attack site) for ~28% of the simulation time. In contrast, $\Delta^1$-imine forms sampled this competent pose much less frequently (<17%).
• Conformational Control: The orientation of the 6$\alpha$-hydroxyethyl group dictates water positioning. Conformation I (~50°) is required to stabilize the deacylating water via hydrogen bonding with both the hydroxyethyl oxygen and KCX73. This conformation is predominantly sampled by the $\Delta^2$-enamine tautomer.
• Product Retention: QM/MM-derived simulations showed that $\beta$-lactone products are retained more stably in the OXA-48 active site than hydrolyzed products. Hydrolyzed meropenem frequently dissociated from the oxyanion hole, whereas $\beta$-lactone products remained bound, consistent with the reversible nature of $\beta$-lactone formation.

C. Mechanism of Enhanced Activity in OXA-519 (Val120Leu)

• Structural Changes: The Val120Leu mutation in OXA-519 opens the "deacylating water channel." Unlike OXA-48, where Val120 (in the t rotamer) blocks solvent access to KCX73, Leu120 in OXA-519 allows a water molecule (W2) to reside deep in the active site near KCX73.
• Enhanced Sampling: OXA-519 acyl-enzymes sample hydrolysis-competent and $\beta$-lactone-promoting conformations significantly more frequently (>33% of trajectories) than OXA-48 (<25%).
• Mechanism of Enhancement:
  1. Hydration Control: In OXA-48, the Val120 side chain must rotate to a rare g+ rotamer to expel a second water molecule that would otherwise inhibit KCX73 activation. In OXA-519, the Leu120 side chain naturally prevents the ingress of this inhibitory second water, keeping the catalytic base more active.
  2. $\beta$-Lactone Promotion: The Leu120 mutation allows the 6$\alpha$-hydroxyethyl group to approach KCX73 more frequently, facilitating the intramolecular recyclization required for $\beta$-lactone formation.
• Crystallographic Evidence: OXA-519 structures captured hydrolyzed products and acyl-enzymes in the $\Delta^2$-enamine form more readily, confirming faster turnover.

4. Significance

• Mechanistic Insight: The study establishes that the $\Delta^2$-enamine tautomer is the catalytically competent form for carbapenem deacylation in Class D SBLs, driven by specific 6$\alpha$-hydroxyethyl conformations that stabilize the deacylating water.
• Role of Hydration: It highlights that the regulation of water access to the catalytic base (KCX73) via the Val120/Leu120 residue is a critical determinant of catalytic efficiency.
• Drug Design Implications:
  • Strategies to restrict the rotation of the 6$\alpha$-hydroxyethyl group (e.g., C5$\alpha$-methyl carbapenems like NA-1-157) could prevent the enzyme from adopting the deacylation-competent conformation.
  • Modifying the carbapenem scaffold to favor the less reactive $\Delta^1$-imine tautomer could inhibit turnover.
  • Understanding the $\beta$-lactone pathway suggests that stabilizing this reversible intermediate could act as a mechanism-based inhibitor.
• Evolutionary Context: The findings explain how a single point mutation (Val120Leu) in the deacylating water channel can drastically alter the catalytic profile of OXA-48, providing a model for how resistance evolves in clinical variants like OXA-519.

In conclusion, the paper demonstrates that the turnover of carbapenems by OXA-48 is a complex interplay of tautomerisation, side-chain dynamics, and active site hydration, where subtle structural changes can shift the balance between slow hydrolysis and rapid degradation via alternative pathways.