Ion-combination specific effects driving the enzymatic activity of halophilic alcohol dehydrogenase 2 from Haloferax volcanii in aqueous ionic liquid solvent mixtures

Biocatalysis in ionic liquids enables novel routes for bioprocessing. Enzymes derived from extremophiles promise greater stability and activity under ionic liquid (IL) influence. Here, we probe the enzyme alcohol dehydrogenase 2 from the halophilic archaeon Haloferax volcanii in thirteen different ion combinations for relative activity and analyse the results against molecular dynamics (MD) simulations of the same IL systems. We probe the ionic liquid property space based on ion polarizability and molecular electrostatic potential. Using the radial distribution functions, survival probabilities and spatial distribution functions of ions, we show that cooperative ion–ion interactions determine ion–protein interactions, and specifically, strong ion–ion interactions equate to higher enzymatic activity if neither of the ions interact strongly with the protein surface. We further demonstrate a tendency for cations interacting with the protein surface to be least detrimental to enzymatic activity if they show a low polarizability when combined with small hydrophilic anions. We also find that the IL ion influence is not mitigated by the surplus of negatively charged residues of the halophilic enzyme. This is shown by free energy landscape analysis in root mean square deviation and distance variation plots of active site gating residues (Trp43 and His273) demonstrating no protection of specific structural elements relevant to preserving enzymatic activity. On the other hand, we observe a general effect across all IL systems that a tight binding of water at acidic residues is preferentially interrupted at these residues through the increased presence of potassium ions. Overall, this study demonstrates a co-ion interaction dependent influence on allosteric surface residues controlling the active/inactive conformation of halophilic alcohol dehydrogenase 2 and the necessity to engineer ionic liquid systems for enzymes that rely on the integrity of functional surface residues regardless of their halophilicity or thermophilicity for use in bioprocessing.


Supplementary Data Description Experimental data
The data points obtained from measuring spectrophotometrically the increase in NADPH are provided in the excel sheet named "Experimental_data_ADH2_IL_ActivityAssays.xlsx".
Columns have been renamed for identification.

MD amber start files
Amber input files for simulations (xxx.prmtop and xxx.inpcrd) are deposited under https://doi.org/10.5281/zenodo.10066549.Additionally, files derived from packmol (xxx_box.pdb)and xLEaP (xxx.pdb,HvADH2net.pdb)are provided for most of the modelled IL systems.The model of tetrameric ADH2 (HvADH2.pdb),which was built on the homology model of the monomer obtained from I-TASSER and which serves as input to the IL systems built with packmol and xLEaP, is provided therein as well.

MD trajectories
Since xxx.mdcrd files were too big to be deposited (some > 50 GB), files were converted into gromacs trajectory files xxx.xtc and xxx.gro using the trajectory converter from the python MDAnalysis suite in Jupyter Notebook.These are deposited under https://doi.org/10.5281/zenodo.4706937.Due to a lack of matching annotations of Zinc coordinating residues in gromacs, structural and catalytic Zinc coordinating CYS and HIS residues (6 per monomer) are missing from converted trajectories.

H-bond data from intra-protein salt-bridges
VMD output .datfiles of salt-bridges occurring for each step of the trajectory are zipped in the file "saltbridges.zip",which is available at https://doi.org/10.5281/zenodo.11916740.

MD Analysis Scripts and exported .csv files
Scripts derived from the python library 'MD Analysis' to calculate RDFs, SPs and RMSDs and the respective data points stored in .csvsheets used to plot RDFs, SPs and RMSDs are zipped in the file "MDAnalysis_Calculated_Plotted_Data.zip".

Cepos InSilico Derived Ion Descriptors
Calculated MEPrange, Polarisability and other ion descriptors in form of .csvfiles as well as .cubefiles and others necessary for visualisation of descriptors are contained in "IonDescriptors.zip".

Parameterised IL ions for MD Simulations
The .lib, .frcmodand .pdbfiles required to parametrise IL ions for simulations are contained in "ILsource.zip".

Ionic liquid descriptor space
The cation [Me 3 S] + was selected based on its tetrahedral structure, which we theorised might be least disruptive to water structuring, and because it shows a remarkably low polarisability and a low range between the most negative and most positive molecular electrostatic potential (in the following referred to as MEP range Hydroxyl-functionalised ILs were of specific interest since the hydroxyl group can act as an additional hydrogen bond donor and acceptor, while carboxylate groups can only act as hydrogen acceptors.[2] This property makes them interesting to investigate, especially with regards to halophilic protein surfaces and the interactions with these. [Choline] + is nontoxic and renewable.It possesses a low polarisability despite its polar hydroxyl group, which is distanced from the not densely charge populated central nitrogen.Its non-hydroxyl counterpart [N 4,1,1,1 ] + , which has similar low polarisability and similar MEP range , was consequently chosen and paired with the medium polarisability and high MEP range anion, [(MeO) 2 OPO] -, with the aim of investigating differences in protein surface interactions.The polarity of hydroxylated cations strongly depends on their respective counter-anion, while their non-hydroxylated versions do not.[3] [Choline] + was hence paired with the monatomic anion

Preparation of ionic liquid mixtures
Following aqueous ionic liquid mixtures were applied:   but becomes even more apparent with the systems where a prolonged interaction exists between ions.

HvADH2
Both ions in IL [P 6,6,6,14 ] + [NTf 2 ] -which interact longest with each other, have a similarly long interaction with the protein surface (see also figure below).In terms of similarity of counter-ion interaction with the protein surface this system is followed by [N show peaks at 2.5 Å and between 5.1-7.1 Å. Radial pairs between K + ions peak at 3.5-5.3Å, 5.7-6.3Å, 6.5-7.3Å and 7.9-8.5Å.
K + associating in its second and/or third shell at 3.7-4.

Me 3 S
Figure S2.While monomer B and monomer D remain static over the course of the trajectories, because monomer B has no cofactor bound and monomer D stays bound to NAD + over the whole course of the trajectory, monomers A and C expel NAD + .In monomer A the cofactor is guided out of the binding pocket between 10 and 20 ns, while in monomer C NAD + is released from the binding pocket at around 100 ns.This reflects well in the diverging distance between residues Asp 155 to His 59 between monomers.Distance between Cys 38 and His 59 stays remarkably constant for all monomers and confirms the overall stability of and reproducibility between monomers.

SI Figure S9 .
figure below).This is not only observed for the systems [DiMIM] + [MeSO 4 ] -and [BMIM] + [MeOEtSO 4 ] -, 3 Å and 4.3-5.8Å, respectively, may allow for the occupation of H 2 O in the fourth hydration shell starting at 4.1 Å consistent with the second association distance between K + and H 2 O of 3.3 Å.The association between K + and H 2 O based on their third interaction distance between 4.5-6.5 Å may give rise to the separation of H 2 O molecules according to their second interaction distance of 5.1-7.1 Å and separates K + ions according to their third interaction distance of 5.1-7.1 Å. Direct association of water to COO -at 1.5-2.1 Å may account for the second coordination shell of K + around COO -at 3.7-4.3Å, in concordance with the first hydration shell of H 2 O around K + of 2.9 Å.The second hydration shell of H 2 O around COO -at 2.9-3.1 Å may account for the third hydration shell of H 2 O to COO -at 3.3-4.1 Å in concordance with the inter H 2 O distance of 2.5 Å. Farther than 6.0 Å from COO -, K + and H 2 O can assume either of their three association distances, which is also in congruence with the 'spikey' profile of K + ions to K + ions association distance between 5.7-6.3Å and 6.5-7.3Å.These distinct peaks merge in bulk water (< 500 Å around COO -).Closer to the protein surface this allows for varying hydration shells of H 2 O around COO -.Further elaboration of how shell distances may explain or fit in with other shell distances in conjunction with SDFs is given in Figure4of the main manuscript.Spatial distribution functions around non-negativelycharged residues SI Figure S15.SDFs of non-acidic residues (A) Thr1073, (B) Lys1064, (C) Gln1070 and (D) Leu280.The polar uncharged side chain of Thr1073 and Gln1070 show residue specific coordination of K + ions.Coordination of K + ions is farthest removed around the positively charged residue Lys1064 and the apolar residue Leu280.However, solvent structure surrounding residues remains unbroken for all four residues.A highly interesting coordination is observed for Threonine, where the tetrahedral structure of the C 4 sidechain atom appears to subject the water structure in the second solvation shell as well as K + ion distribution to a coordination maintaining the character of a triad, in case of K + a double triad.
).In order to cover a wide experimental space, this cation was paired with a monatomic anion showing low polarisability and a low MEP range ([I] -), a water-miscible anion having medium polarisability and a high MEP range ([MeSO 4 ] -), and a hydrophobic anion showing high polarisability and a high MEP range ([NTf 2 ] -).To further cover for low polarisability, but in conjunction with a high MEP range on part of the cation, [Me 3 SO] + was combined with[Iodide] -, and to cover for a high polarisability and high MEP range on part of the cation, [P 6,6,6,14 ] + was combined with the anion [NTf 2 ] -, yielding a biphasic system.Sulfuric acid-based ionic liquids are inexpensive, easily recyclable and efficient catalysts in a range of chemical syntheses.[1]Especially with regards to deriving precursors from biomass or processing these precursors further to obtain pharmaceuticals or fine chemicals, these protic ILs are an interesting class to be investigated towards their impact on protein structural integrity and enzymatic activity.In order to investigate a range of emergent properties, the sulfuric acid derivative [MeSO 4 ] -was further combined with a water-immiscible cation of high polarisability and medium MEP range [N 1,1,8,8 ] + , yielding a second bi-phasic system, as well as with a charge-dispersed imidazolium cation [DiMIM] + .This latter cation shows similar low polarisability and MEP range as [Me 3 S] + and was chosen to interrogate the influence of the structure by comparing the tetrahedral structure of [Me 3 S] + to the planar structure of [DiMIM] + .A second sulfuric acid derivate, which was additionally ether-functionalised,

distribution functions of K + and O w around C/C of Glu/Asp residues, Gln/Asn residues and between K + -K + , K + -O w and O w -O w SI Figure S13.
RDFs obtained from MD simulation of the native HvADH2 system for K + and H 2 O.