Lactonase Lab
Follow one enzyme, the lactonase SsoPox, as it opens the ring of a bacterial signal molecule. Press Play, or go step by step.
Part 1 · The mechanism
One turnover, step by step
Signal drawn: 3-oxo-C12-AHL, L form. Schematic, not to scale. The animation is not in real time. Swipe the drawing sideways to see the acyl chain.
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The enzyme at rest
Two metal ions sit 3.3 to 3.5 Å apart at the bottom of the active site: iron in the buried α site, cobalt in the more exposed β site. Four histidines, an aspartate and a carboxylated lysine (Lys137) hold them. The carbamate of Lys137 bridges both metals, its negative charge shared by its two oxygens. A water molecule bridges the two metals, which act as a Lewis acid and turn it into a hydroxide ion: the nucleophile of the reaction.
Based on crystal structures of SsoPox. That the bridging water is a hydroxide is proposed.
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The signal binds
SsoPox takes the L form of the signal, the one bacteria make. The drawing shows it for 3-oxo-C12-AHL: at the ring carbon that carries the amide, the nitrogen points behind the plane (hashed bond).
The lactone ring settles on the two metals, positioned by Trp263. Its carbonyl oxygen points to the cobalt and takes a hydrogen bond from Tyr97. Its ring oxygen faces the iron. The carbonyl carbon ends up 2.6 to 2.9 Å from the bridging hydroxide, and the acyl chain lies in a hydrophobic channel.
Based on crystal structures of SsoPox, listed in the sources.
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The transition state
‡ Highest point of the reaction
Held on the metals, the carbonyl carbon is more electrophilic, and the hydroxide attacks it. At the transition state the new bond between the hydroxide oxygen and the carbon is half formed, the carbon becomes tetrahedral and the negative charge moves onto the carbonyl oxygen, where the β metal and Tyr97 stabilize it.
Proposed from the structures.
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The ring opens
Signal degraded
The attack ends in a tetrahedral intermediate: the carbon holds four bonds and its former carbonyl oxygen carries a full negative charge. The electron pair on that oxygen then folds back and the C=O bond forms again. The bond between the carbon and the ring oxygen breaks instead. The ring is open: one end is a carboxylic acid, the other an alcoholate. From here on the molecule is no longer a signal.
Proposed from the structures.
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Protons settle
The alcoholate takes a proton and becomes the alcohol of homoserine. Which group gives that proton is not established: an aspartate that binds the α metal (Asp256 in SsoPox) has been proposed for other lactonases. The new acid gives its own proton to the water. This released proton is what the assay measures.
Not established: the proton donor. Measured: the acidification that follows ring opening, with the pH indicator cresol purple.
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The signal is degraded
Signal degraded
The open-ring product, an N-acyl homoserine, leaves the active site. It no longer activates the signal receptor, so the message is lost. A new water molecule takes the bridging place and the enzyme is ready for the next signal. The gold oxygen came from water and now belongs to the product.
Proposed. The order in which the product leaves and water returns is not known.
Reading the drawing
- Oxygen that comes from water
- Other oxygens
- Nitrogen
- Bond pointing back, with (S): the L form of the signal
- Dashed bond: partial, forming, breaking or shared
- Electron pair on the move
- Contact, with the distance in the crystal
Part 2 · The kinetics
Many turnovers
One enzyme molecule repeats this cycle again and again. Watch many enzyme molecules degrade 3-oxo-C12-AHL, following the Michaelis–Menten equation with the constants measured for SsoPox. Then see what one engineered mutation, W263I, changes.
- Ring closed: active signal
- Ring open: degraded
- Time
- Signal left
- Half is degraded after
- Rings opened by each enzyme at the start
Constants used for 3-oxo-C12-AHL
| SsoPox | kcat (s⁻¹) | KM (µM) | kcat/KM (M⁻¹ s⁻¹) |
|---|---|---|---|
| Wild type | 1.01 ± 0.13 | 456 ± 128 | 2.22 (± 0.68) × 10³ |
| W263I | 1.80 ± 0.05 | 17.8 ± 4.9 | 1.01 (± 0.28) × 10⁵ |
SsoPox with cobalt, at 25 °C and pH 8.3 (Hiblot and others, 2013). In W263I, tryptophan 263 is replaced by isoleucine. SsoPox takes the L form of the signal, the one bacteria make.
What the lab leaves out
- The mechanism is a proposal. It rests on crystal structures of the enzyme with a bound analogue, not on a direct view of the reaction.
- The curve is a model. It assumes far more signal than enzyme, a stable enzyme and no effect of the product.
Sources
- Jacquet P, Billot R, Shimon A, Hoekstra N, Bergonzi C, Jenks A, Chabrière E, Daudé D, Elias MH. Changes in Active Site Loop Conformation Relate to the Transition toward a Novel Enzymatic Activity. JACS Au 4(5):1941-1953 (2024). Recent crystal structures of SsoPox variants.
- Hiblot J, Gotthard G, Elias MH, Chabrière E. Differential active site loop conformations mediate promiscuous activities in the lactonase SsoPox. PLoS One 8:e75272 (2013). Kinetic constants.
- Crystal structures of SsoPox in the Protein Data Bank: 2VC5, 2VC7, 3UF9, 4KER, 4KES, 4KET, 4KEU, 4KEV, 4KEZ, 4KF1, 5VRI, 5VRK, 5VSA, 5W3U, 5W3W, 5W3Z, 8SF2, 8SF9, 8SFA, 8SFB, 8SFC, 8SFD, 8SFK, 8SFM.
Read more about our work on bacterial communication, or try Quorum Lab to see what the signal does before it is cut.