Enzyme Multifunctionality by Control of Substrate Positioning Within the Catalytic Cycle—A QM/MM Study of Clavaminic Acid Synthase

Abstract

Clavaminic acid synthase from Streptomyces clavuligerus is an FeII/2‐oxoglutarate‐dependent dioxygenase, crucial for the biosynthesis of the β‐lactamase inhibitor clavulanic acid. It catalyses three consecutive oxidative reactions, that is, hydroxylation, cyclisation and desaturation, in a single binding cavity. As follows from the results of this QM/MM study, CAS versatility and selectivity depends on the binding cavity, which interplays differently with the substrate for each reaction. The enzyme–substrate interactions affect the substrate's ability to re‐position during the reaction, either constraining it in its primary position, which impedes processes other than oxygen rebound, or allowing change, which facilitates desaturation. This differential effect originates from two aspartate residues, which strongly interact with the guanidine group of the hydroxylation substrate and stabilise the orientation of the molecule. These residues interact less effectively with the smaller amine group of the desaturation substrate(s), aiding their re‐positioning and the subsequent formation of a double bond.

Properties of the Reactants and Their Interactions within and with the Enzyme Binding Cavity Determine Reaction Selectivities. The Case of Fe(II)/2‐Oxoglutarate Dependent Enzymes

“…The course of the reaction can be also controlled by charged residues present in the second coordination sphere of Fe(IV). Charge‐charge interactions can govern the (regio)selectivity of the reaction, according to the principle of negative catalysis, as demonstrated in QM/MM studies on hyoscyamine 6β‐hydroxylase (H6H) and clavaminic acid synthase (CAS) [36,163] . For the former enzyme, the regioselectivity of hydroxylation comes from electrostatic repulsion between the lysine side chain and the charged amine group of the tropane ring of the substrate.…”

“…The desaturation reaction mechanisms also involve change of conformation of the radical, yet it is not associated with an increase of ΔG (ΔE ONIOM ) barrier, as the amino group does not interact strongly with aspartate residues – the interactions are mediated by water molecules. Hence, in CAS, the ability of the substrate‐derived radical to re‐position with respect to the Fe‐binding site facilitates desaturation, whereas conservation of its position (due to attractive electrostatic interactions) promotes hydroxylation [163] . The charge distribution within the cavity can not only modulate the ability of the substrate to reposition, but also differentiate the strengths of the C−H bonds of the substrate as described in QM studies on VioC.…”

“…The oxidation of substrate involves two one‐electron processes; typically the barrier for the second one (quenching of the radical) is sufficiently high (typically in the range of 4–11 (max. 20) kcal mol −1 ) to justify treating the two steps as independent and analysing stabilising/destabilising factors for each step separately [36,115,127,143,163,164,166,170–172] . However, from the experimental results it emerges that reaction rate (determined by HAT) and the rate of radical‐rebound are positively correlated and depend on the product selectivity of the reaction, that is, hydroxylation is frequently associated with faster HAT than any alternative reaction.…”

“…[82] Some ODDs exhibit ring formation activity, and here the reaction follows an analogous mechanism to desaturation with two consecutive HATs, or HAT and a subsequent ET/PT. In cyclisation the second HAT step proceeds with a different [77] IsnB/AmbI3 [78,79] carbocation formation FLS, [81] VioC* [77,82] specific order of CÀ H bond cleavage CAS, [29,163] AusE/PrhA double mutant, [35,164] CarC [127] re-position of the radical cyclisation ODD from the biosynthesis pathway for (À )-podophyllotoxin [89] carbocation formation DabC, [90] SnoK, [91] KabC [92] attack of the C radical at a double bond oxacyclisation/epoxidation CAS [104] , H6H [105] , LolO [106] specific conformation of the substrate or its coordination to Fe(IV)/Fe(III) AsqJ, [119] AlkB, [120][121][122] LdoA*, [123] T7H* [69,124,125] addition of O to the double bond (instead of initial HAT) ring rearrangement DAOCS [93] attack of the S radical at the electron-rich system PrhA and AusE, [35,164] EasH [101][102][103] attack of the C radical at a double bond endoperoxidation FtmOx1, [129,130] NvfI [131,132] use of additional molecule of dioxygen halogenation carrier dependent: CytC3, [167] SyrB2, [134,135] HctB <...>…”

“…Charge-charge interactions can govern the (regio)selectivity of the reaction, according to the principle of negative catalysis, as demonstrated in QM/MM studies on hyoscyamine 6β-hydroxylase (H6H) and clavaminic acid synthase (CAS). [36,163] For the former enzyme, the regioselectivity of hydroxylation comes from electrostatic repulsion between the lysine side chain and the charged amine group of the tropane ring of the substrate. This destabilising effect increases the ΔG (ΔE ONIOM ) barrier for one of the intrinsically equivalent channels for HAT.…”