Photon Factory Activity Report 2019 #37 (2020)

BL-1A, -5A, -17A, AR-NW12A/2017G514

Crystal structure analysis of the substrate-binding complexes ofPET degrading enzyme Cut190
Nobutaka NUMOTO1,*
1
Medical Research Institute, Tokyo Medical and Dental University (TMDU),
1-5-45 Yushima Bunkyo-ku, Tokyo, 113-8510, Japan
1 Introduction
Cutinase belongs to the lipase family, and some of thecutinase or cutinase-like enzyme have been reported that it
can degrade polyethylene terephthalate (PET), which is a
representative of persistent plastics. A cutinase from athermophilic isolate, Saccharomonospora viridis AHK190(Cut190) show high thermostability and enzymatic activity,
and therefore it would provide attractive solution forrecycling of waste plastic by the enzymatic treatment.
We have previously reported that Cut190 retain theactivity at a temperature higher than the glass transition
temperature of PET of approximately 60–65 °C. This highthermostability is indispensable for efficient degradationof PET. We have also characterized remarkable nature of
Cut190 that calcium ions regulate the thermostability andenzymatic activity. Crystal structures of calcium boundand free form of Cut190 [1] have demonstrated thatcalcium binding site is far from the active site and thecalcium ion regulates open/closed conformationaltransition at the active site as an allosteric effector.
In this study, we used improved mutant of Cut190
S226P/R228S, whose thermostability and enzymatic
activity are enhanced. We introduced additional inactive
substitution at the active Ser residue as S176A, forpreparing the substrate complexes of Cut190. We havesuccessfully solved two small substrate bound states ofCut190 S176A/S226P/R228S [2]. The obtained structures
allow us to discuss more detailed mechanism of theenzymatic activity regulated by calcium binding anddissociating.
2 Experiment
Cut190S176A/S226P/R228S
mutantwasoverexpressed in Escherichia coli and purified as
described previously. Crystallization was performed by thehanging-drop vapor-diffusion method at 20ºC. Prior to datacollection, the crystals were soaked in cryo-protectant
solutions containing 20% (v/v) glycerol and 10-100 mM
substrates of monoethyl succinate (Et-succinate) andmonoethyl adipate (Et-adipate), along with their respectivereservoir buffers and flash-frozen using liquid nitrogen.
X-ray diffraction experiments were performed at thebeamlines BL-1A, -5A, -17A, and NW12A at PF and PFAR, KEK. All data were processed and scaled using XDS[3]. The initial phases were determined by the molecularreplacement method using the program PHASER [4].
Several cycles of manual model rebuilding by using COOT[5] and refinement by using PHENIX [6] were performed.

3 Results and Discussion
We obtained the crystals suitable for the substrates
soaking with the buffer containing 100 mM MES (pH 6.5),10 mM zinc sulfate heptahydrate, and 25% (v/v) PEG
monomethyl ether 550.
The structure of Et-succinate bound form wasdetermined at 1.34 Å resolution, and clearly indicate thatthe enzyme release calcium ion and conformational change
allow to accommodate the substrate tightly, thereby it isassumed as a pre-reaction state (Fig. 1, left). In contrast,
Et-adipate bound form was determined at 1.4 Å resolution,and is assumed to be a post-reaction state (Fig. 1, right)
because the carboxyl group of the bound substrate is
located proximally to the active site, mimicking just aftercleavage at the ester group of the substrates.
We propose the novel enzymatic cycle of Cut190 [2].
First, the calcium ion at the allosteric site temporarily
dissociates and Cut190 shifts to the pre-reaction state when
the substrate is bound at the active site. Next, the enzymatic
reaction proceeds to cleave the ester group, and the calcium
ion is bound again after the cleavage of the substrate. The
re-binding of Ca causes the structural changes to promote
product elimination and new substrate binding.

Fig. 1: Structures of pre-reaction (cyan) and post-reaction(yellow) states of Cut190 S176A/S226P/R228S.
References[1] T. Miyakawa et al., Appl. Microbiol. Biotechnol. 99,4297 (2015).
[2] N. Numoto et al., Biochemistry 57, 5289 (2018).
[3] W. Kabsh, Acta Cryst. D66, 125 (2010).
[4] A. J. McCoy et al., J. Appl. Crystallogr. 40, 658 (2007).
[5] P. Emsley et al., Acta Cryst. D66, 486 (2010).
[6] P. D. Adams et al., Acta Cryst. D66, 213 (2010).
* numoto.str@mri.tmd.ac.jp