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. 2022 Nov 30;13(1):7379.
doi: 10.1038/s41467-022-35029-3.

Time-resolved β-lactam cleavage by L1 metallo-β-lactamase

M Wilamowski  1   2   3 , D A Sherrell  4 , Y Kim  1   4 , A Lavens  4 , R W Henning  5 , K Lazarski  4 , A Shigemoto  6 , M Endres  1 , N Maltseva  1 , G Babnigg  1 , S C Burdette  6 , V Srajer  5 , A Joachimiak  7   8   9
Affiliations

Time-resolved β-lactam cleavage by L1 metallo-β-lactamase

M Wilamowski et al. Nat Commun. .

Abstract

Serial x-ray crystallography can uncover binding events, and subsequent chemical conversions occurring during enzymatic reaction. Here, we reveal the structure, binding and cleavage of moxalactam antibiotic bound to L1 metallo-β-lactamase (MBL) from Stenotrophomonas maltophilia. Using time-resolved serial synchrotron crystallography, we show the time course of β-lactam hydrolysis and determine ten snapshots (20, 40, 60, 80, 100, 150, 300, 500, 2000 and 4000 ms) at 2.20 Å resolution. The reaction is initiated by laser pulse releasing Zn2+ ions from a UV-labile photocage. Two metal ions bind to the active site, followed by binding of moxalactam and the intact β-lactam ring is observed for 100 ms after photolysis. Cleavage of β-lactam is detected at 150 ms and the ligand is significantly displaced. The reaction product adjusts its conformation reaching steady state at 2000 ms corresponding to the relaxed state of the enzyme. Only small changes are observed in the positions of Zn2+ ions and the active site residues. Mechanistic details captured here can be generalized to other MBLs.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Experimental set-up for TR-SSX at 14-ID-B BioCARS beamline.
a Polychromatic x-ray beam depicted with simplified representation of pulse train exposure, containing 24 x-ray pulses with duration of 100 ps each and total pulse train duration of 3.7 µs. ALEX chip moves during scan in a meander-pattern over the preset area of the chip containing slurry of immobilized microcrystals. Laser pulse is coordinated with release of x-ray beam by a time delay that corresponds to a specific time point of reaction. The typical Laue diffraction pattern is shown obtained from L1 MBL crystal during TR-SSX experiment. b Movement of the ALEX chip across the Y-axis with step size labeled as blue bars with an overlaid red baseline that indicates the diameter of a laser pulse spot, source data is available as Source data file. c Scheme of fixed-target SSX data collection on nylon mesh crystals holder utilizing the pump-probe system. The X and Y axis step sizes of the chip are labeled as red arrows. SSX data collection were conducted as pump-probe system with specific time-delay between laser pulse and a train of 100 ps pink-beam x-ray pulses. To avoid the impact of zinc diffusion on data collection the step size of the SSX crystal holder was increased for the detection of longer time-points of moxalactam cleavage reaction. d Microscope photography of x-ray burn paper after test run of pump-probe SSX system.
Fig. 2
Fig. 2. Overview of crystal structure of L1 from S. maltophilia in a complex with hydrolyzed moxalactam.
a Structure of L1 is shown as cartoon (salmon), active site contains moxalactam (green sticks), and two zinc ions are showed in yellow. Bottom surface depicts the Coulombic electrostatic potential plotted on the surface of the L1 in complex with moxalactam at 295 K (electrostatic potential calculated using default settings in UCSF Chimera). b Cartoon representation of L1 MBL superposed with crystal structures of B1, B2, and B3 subclasses of MBLs depicted as licorice models. Crystal structure of L1 MBL determined at room temp using SSX (PDB entry 7L52 - magenta) was superimposed with structures of MBL representatives: B1 subclass NDM-1 from K. pneumoniae (PDB entry 6TWT - cyan), B2 subclass mono-zinc MBL SFH-I from Serratia fonticola (PDB entry 3SD9 - yellow), B3 subclass FEZ-1 MBL from Legionella gormanii (PDB entry 5WCK - gray), two zinc ions are showed in pink. c Comparison of key residues in the active site of MBL subclasses. Zinc atoms showed as pink spheres, water molecules are depicted as red spheres with a small radius.
Fig. 3
Fig. 3. TR-SSX structures of moxalactam bound to the active site of L1 MBL.
a Schematics of moxalactam hydrolysis and atom numbering scheme. b Room temperature SSX and TR-SSX structures of L1 active site representing different states (with one zinc, with no metal (both EDTA treated), with one zinc ion bound “dark” structure, time points from 20 to 4000 ms and “pink” reference with two zinc ions and fully hydrolyzed moxalactam). His263 coordinating Zn2 is not displayed for clear view of moxalactam. The 2Fo-Fc map (blue mesh) contoured at 1.0 σ level (carved at 1.4 Å) around moxalactam and zinc ions in catalytic site of L1 MBL. c Substrate, intermediate and product of moxalactam hydrolysis by L1 are shown at three time points. Representation of three key steps captured during reaction (100 ms bound uncleaved moxalactam – substrate (green), 150 ms hydrolyzed moxalactam intermediate (orange), 2000 ms structure with hydrolyzed moxalactam after reaction – product (aqua)). Legend on left site of image illustrates shades of pink spheres that visualize zinc atoms.
Fig. 4
Fig. 4. TR-SSX crystal structures of moxalactam of the active site of L1 MBL.
a L1 active site structure at 150 ms with hydrolyzed moxalactam (in yellow-red-blue), zinc (magenta) and protein residues (in silver-blue-red). b Difference electron density map calculated as Fobs(150 ms)-Fobs(100 ms). The differential map was calculated with Phenix isomorphous difference maps module. Negative electron density showed as red mesh, positive as green mesh. Depicted in the Coot electron density map are countered with a 1.9 σ level. Orange lines illustrate cleaved moxalactam at 150 ms, green lines represent uncut moxalactam at 100 ms. c Distances between atoms key in description of L1 induced β-lactam cleavage reaction (rainbow). d Distances in TR-SSX structures of L1 MBL relative to atom positions in 20 ms crystal structure of L1 MBL (illustrated as a heat-map). e TR changes in distances between zincs and moxalactam atoms modeled in L1 active site, source data is available as Source data file. f Evolution of electron density during cleavage of moxalactam by L1 MBL. The 2Fo-Fc map contoured at 1.0 σ level (carved at 1.4 Å) around moxalactam; maps were depicted as olive mesh for 100 ms, blue mesh for 150 ms, and black mesh for 2000 ms. g Catalytic mechanism proposed for L1 MBL based on TR-SSX experiments.
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