Structures of lactaldehyde reductase, FucO, link enzyme activity to hydrogen bond networks and conformational dynamics

Abstract

A group-III iron containing 1,2-propanediol oxidoreductase, FucO, (also known as lactaldehyde reductase) from Escherichia coli was examined regarding its structure-dynamics-function relationships in the catalysis of the NADH-dependent reduction of (2S)-lactaldehyde. Crystal structures of FucO variants in the presence or absence of cofactors have been determined, illustrating large domain movements between the apo and holo enzyme structures. Different structures of FucO variants co-crystallized with NAD + or NADH together with substrate further suggest dynamic properties of the nicotinamide moiety of the coenzyme that are important for the reaction mechanism. Modelling of the native substrate (2S)lactaldehyde into the active site can explain the stereoselectivity exhibited by the enzyme, with a critical hydrogen bond interaction between the (2S)hydroxyl and the side-chain of N151, as well as the previously experimentally demonstrated pro-(R) selectivity in hydride transfer from NADH to the aldehydic carbon. Furthermore, the deuterium kinetic isotope effect of hydride transfer suggests that reduction chemistry is the main rate-limiting step for turnover which is not the case in FucO catalysed alcohol oxidation. We further propose that a water molecule in the active sitehydrogen bonded to a conserved histidine (H267) and the 2 0 -hydroxyl of the coenzyme ribosefunctions as a catalytic proton donor in the protonation of the product alcohol. A hydrogen bond network of water molecules and the side-chains of amino acid residues D360 and H267 links bulk solvent to this proposed catalytic water molecule.

Crystal structures and kinetic studies of a laboratory evolved aldehyde reductase explain the dramatic shift of its new substrate specificity

“…The pH dependency of k cat and k cat /K M in the reduction of compound 3 is relatively flat (Fig. S3), as observed for the wild-type enzyme for the reduction of compound 1 (Blikstad & Widersten, 2010), which is in agreement with the proposed reaction mechanism, whereby a catalytic water molecule provides the proton for the reduction reaction (Zavarise et al, 2023).…”

“…In the FucO structures of the point-mutation variants reported here, as well as in previous work (Zavarise et al, 2023), the active sites are complexed with Fe 2+ , like in the wild-type 2bl4 structure (Montella et al, 2005), and the Fe 2+ position is the same in all these structures. In the 1rrm and 5br4 crystal structures this Fe 2+ is replaced by Zn 2+ .…”

“…In the complexes obtained by cocrystalization with the nucleotides NADH or NAD + , the density of the ADP-ribose part of the nucleotide was always clearly defined by the electron density map, whereas the electron density for the nicotinamide moiety was usually weaker, suggesting that either the nicotinamide ring is partially disordered, or NAD + or NADH is partially hydrolysed, or both. In the structures with poor density for the nicotinamide ring the nucleotide was modeled as ADP-ribose (ADPR), as also described previously (Zavarise et al, 2023).…”

“…A5 catalyzes the reduction of its physiological substrate (compound 2) with a similar k cat value as the wild-type, but it requires higher substrate concentrations to reach saturation (K M of compound 2 has increased 100-fold; Table 2). The substantial increase in K M for compound 2 reflects the importance of the acetamide side chain of Asn151, being hydrogen bonded with the 2-hydroxyl moiety of compound 2 (Zavarise et al, 2023). However, the activity with the more bulky substrate, compound 3, as judged by k cat /K M , has increased by 150-fold with respect to the wild-type (Table 2).…”

“…The pyrophosphate moiety of NAD is bound at the N-terminal end of the -helix starting with Gly97. The catalytic site, near the bound nicotinamide moiety of NAD, is shaped by regions of both domains and recent structural studies have shown that the subunit can exist in both open and closed states in which the relative position of these two domains with respect to each other is different (Zavarise et al, 2023). It is proposed that in the closed state the competent ternary complex is formed.…”