Further analysis of the US trajectories reveals that hydration around Lys73:OQ2 correlates with the rotamer of Val120. Valine has three rotamers for the χ1 dihedral (N-Cα-Cβ-Cγ1): the g+ rotamer around 50°, t around 180°, and g-around 300° (Figure , Figure ). In the starting structures for simulations, Val120 is in the t orientation for both carbapenems (for meropenem, partial occupancy for both t and g-rotamers was observed in the deposited structure, but only the t rotamer was used in the computational model building). The rotameric state can switch to either g+ or g-during MD simulations (Figure ). For the g+ rotamer, one of the methyl groups points directly towards Lys73, which only leaves space for a single water molecule next to Lys73:OQ2; this water is positioned to accept a hydrogen bond from Gln124 and to donate one to Lys73. Conversely, the t rotamer allows for a second water molecule to occupy the space between Lys73 and Val120, and this water molecule is able to donate hydrogen bonds to both Lys73:OQ2 and the Val120 backbone carbonyl. Val120 is part of motif II, which is formed by residues Ser118 -Val120 and is conserved across class D β-lactamases. Together with Leu158, it forms the so-called 'deacylating water channel' in the vicinity of Lys73; this hydrophobic patch partly shields the active site from bulk solvent. For other OXA enzymes, a similar water channel has been proposed to open upon substrate binding to allow for water ingress into the active site and therefore for efficient deacylation. For OXA-48, previous comparison of apoenzyme and acylenzyme structures shows that substrate binding shifts Val120 and Leu158 only slightly, and that the water channel is more open than e.g. in OXA-23. Access of water into the catalytic position next to the substrate and Lys73 is necessary for antibiotic hydrolysis, but as we indicate above, any additional solvent in the active site will impair reactivity. In OXA-48, it appears that Val120 (and the specific rotamers that it samples) is an important gateway residue controlling approach of bulk solvent to Lys73:OQ2. Our previous work (on ceftazidime hydrolysis in OXA-48-like enzymes) indicates that Leu158 modulates hydration around Lys73:OQ1. Notably, Val120 is mutated to a leucine in OXA-519, a single point mutant of OXA-48; this mutation results in an increase in measured hydrolysis for some 1β-methyl carbapenems, such as meropenem and ertapenem, but decreased imipenemase activity. Compared to OXA-48, OXA-519 also increases the proportion of β-lactone reaction products, rather than conventionally formed ring-opened species, hydrolysis products of meropenem. Further, the Val120Leu mutation increases both kcat and KM for meropenem, indicating opposite effects on binding and hydrolysis. The exact effect of the Val120Leu mutation on carbapenem hydrolysis on the molecular level is therefore complex and remains to be determined.