Synthesis of fucosyllactose using α-L-fucosidases GH29 from infant gut microbial metagenome.

Fucosyl-oligosaccharides (FUS) provide many health benefits to breastfed infants, but they are almost completely absent from bovine milk, which is the basis of infant formula. Therefore, there is a growing interest in the development of enzymatic transfucosylation strategies for the production of FUS. In this work, the α-L-fucosidases Fuc2358 and Fuc5372, previously isolated from the intestinal bacterial metagenome of breastfed infants, were used to synthesize fucosyllactose (FL) by transfucosylation reactions using p-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as donor and lactose as acceptor. Fuc2358 efficiently synthesized the major fucosylated human milk oligosaccharide (HMO) 2'-fucosyllactose (2'FL) with a 35% yield. Fuc2358 also produced the non-HMO FL isomer 3'-fucosyllactose (3'FL) and traces of non-reducing 1-fucosyllactose (1FL). Fuc5372 showed a lower transfucosylation activity compared to Fuc2358, producing several FL isomers, including 2'FL, 3'FL, and 1FL, with a higher proportion of 3'FL. Site-directed mutagenesis using rational design was performed to increase FUS yields in both α-L-fucosidases, based on structural models and sequence identity analysis. Mutants Fuc2358-F184H, Fuc2358-K286R, and Fuc5372-R230K showed a significantly higher ratio between 2'FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes after 4 h of transfucosylation. The results with the Fuc2358-F184W and Fuc5372-W151F mutants showed that the residues F184 of Fuc2358 and W151 of Fuc5372 could have an effect on transfucosylation regioselectivity. Interestingly, phenylalanine increases the selectivity for α-1,2 linkages and tryptophan for α-1,3 linkages. These results give insight into the functionality of the active site amino acids in the transfucosylation activity of the GH29 α-L-fucosidases Fuc2358 and Fuc5372. KEY POINTS: Two α-L-fucosidases from infant gut bacterial microbiomes can fucosylate glycans Transfucosylation efficacy improved by tailored point-mutations in the active site F184 of Fuc2358 and W151 of Fuc5372 seem to steer transglycosylation regioselectivity.

The E2 state of FeMoco: Hydride Formation versus Fe Reduction and a Mechanism for H2 Evolution.

The iron-molybdenum cofactor (FeMoco) is responsible for dinitrogen reduction in Mo nitrogenase. Unlike the resting state, E0 , reduced states of FeMoco are much less well characterized. The E2 state has been proposed to contain a hydride but direct spectroscopic evidence is still lacking. The E2 state can, however, relax back the E0 state via a H2 side-reaction, implying a hydride intermediate prior to H2 formation. This E2 →E0 pathway is one of the primary mechanisms for H2 formation under low-electron flux conditions. In this study we present an exploration of the energy surface of the E2 state. Utilizing both cluster-continuum and QM/MM calculations, we explore various classes of E2 models: including terminal hydrides, bridging hydrides with a closed or open sulfide-bridge, as well as models without. Importantly, we find the hemilability of a protonated belt-sulfide to strongly influence the stability of hydrides. Surprisingly, non-hydride models are found to be almost equally favorable as hydride models. While the cluster-continuum calculations suggest multiple possibilities, QM/MM suggests only two models as contenders for the E2 state. These models feature either i) a bridging hydride between Fe2 and Fe6 and an open sulfide-bridge with terminal SH on Fe6 (E2 -hyd) or ii) a double belt-sulfide protonated, reduced cofactor without a hydride (E2 -nonhyd). We suggest both models as contenders for the E2 redox state and further calculate a mechanism for H2 evolution. The changes in electronic structure of FeMoco during the proposed redox-state cycle, E0 →E1 →E2 →E0 , are discussed.

Computational Mechanistic Study of [MoFe3S4] Cubanes for Catalytic Reduction of Nitrogenase Substrates.

Molybdenum-dependent nitrogenase is the most active biological catalyst for dinitrogen reduction. This reaction is catalyzed by a [MoFe7S9C] cofactor (FeMoco). FeMoco can be described as a double-cubane, with [MoFe3S3] and [Fe4S3] parts, bound via an interstitial carbide and three bridging sulfides. Model compounds have been synthesized since early studies of the enzyme and Coucouvanis and co-workers demonstrated that [MoFe3S4] cubanes are active catalysts for many substrates catalyzed by nitrogenase. These reactions include hydrazine reduction to ammonia and cis-dimethyldiazene reduction to methylamine. Experiments implicated molybdenum as the binding site but the mechanisms have not been studied by theoretical calculations before. Here we present a DFT study of the catalytic reaction mechanisms of hydrazine and cis-dimethyldiazene reduction with a [MoFe3S4] cubane. Like in the experiments, molybdenum is revealed as the likely substrate binding site, likely due to the labile ligand on Mo. For the hydrazine mechanism, a reduction event is centered on Fe, specifically on the Fe antiferromagnetically coupled to the mixed-valence pair. After protonation of the distal hydrazine nitrogen, the N-N bond can be cleaved to yield NH3 and a Mo-bound -NH2 intermediate. This is followed by another protonation/reduction step to give an -NH3 intermediate, and finally substituted by the substrate to complete the cycle. The computed mechanisms shed light on the bimetallic cooperativity in these systems where the reduction steps are localized on Fe while the substrate binds to Mo and the reductions require only a free coordination site (on Mo) and a favorable reduction event (to Fe). Although both substrates easily displace the weakly bound acetonitrile ligand, one reduction event is required for hydrazine activation and N-N bond cleavage to give an integer-spin -NH2 intermediate. An integer-spin -NH2 intermediate has been observed as a common intermediate for the enzyme reduction of hydrazine and diazene, suggesting a possible link to the enzyme chemistry.

A model for dinitrogen binding in the E4 state of nitrogenase.

Molybdenum nitrogenase is one of the most intriguing metalloenzymes in nature, featuring an exotic iron-molybdenum-sulfur cofactor, FeMoco, whose mode of action remains elusive. In particular, the molecular and electronic structure of the N2-binding E4 state is not known. In this study we present theoretical QM/MM calculations of new structural models of the E4 state of molybdenum-dependent nitrogenase and compare to previously suggested models for this enigmatic redox state. We propose two models as possible candidates for the E4 state. Both models feature two hydrides on the FeMo cofactor, bridging atoms Fe2 and Fe6 with a terminal sulfhydryl group on either Fe2 or Fe6 (derived from the S2B bridge) and the change in coordination results in local lower-spin electronic structure at Fe2 and Fe6. These structures appear consistent with the bridging hydride proposal put forward from ENDOR studies and are calculated to be lower in energy than other proposed models for E4 at the TPSSh-QM/MM level of theory. We critically analyze the DFT method dependency in calculations of FeMoco that has resulted in strikingly different proposals for this state. Importantly, dinitrogen binds exothermically to either Fe2 or Fe6 in our models, contrary to others, an effect rationalized via the unique ligand field (from the hydrides) at the Fe with an empty coordination site. A low-spin Fe site is proposed as being important to N2 binding. Furthermore, the geometries of these states suggest a feasible reductive elimination step that could follow, as experiments indicate. Via this step, two electrons are released, reducing the cofactor to yield a distorted 4-coordinate Fe2 or Fe6 that partially activates N2. We speculate that stabilization of an N2-bound Fe(i) at Fe6 (not found for Fe2 model) via reductive elimination is a crucial part of N2 activation in nitrogenases, possibly aided by the apical heterometal ion (Mo or V). By using protons from the sulfhydryl group (to regenerate the sulfide bridge between Fe2 and Fe6) and the nearby homocitrate hydroxy group, we calculate a plausible route to yield a diazene intermediate. This is found to be more favorable with the Fe6-bound model than the Fe2-bound model; however, this protonation is uphill in energy, suggesting protonation of N2 might occur later in the catalytic cycle or via another mechanism.

QM/MM calculations reveal a bridging hydroxo group in a vanadium nitrogenase crystal structure.

A new 1.2 Å crystal structure of vanadium nitrogenase, isolated under turnover conditions, recently revealed a light atom ligand (OH or NH) replacing the bridging S2B sulfide of the FeV cofactor. QM/MM calculations on the new structure now reveal the light-atom ligand to be a bridging hydroxo group, probably derived from water binding to the cofactor.