**1. Introduction**

YqhD is a homo-dimeric, NADP(H)-dependent *E. coli* oxidoreductase identified in 2003 [1]. Since its discovery, it has received considerable attention for bioengineering efforts [2], being targeted for its utility in biomass conversion to biofuels [3] and chemical feedstocks [4]. The enzyme was initially identified during the *E. coli* structural genomics program and was shown to be widely distributed throughout the bacteria kingdom [5]. YqhD enzyme works as a homodimer with Zn2+ in the active site, catalyzing the interconversion of alcohols and aldehydes with NADP and NADPH, respectively, as a cofactor. Although YqhD binds Zn2+, it has structural similarities to the iron-dependent (group III) alcohol dehydrogenase enzymes [6]. Initially, YqhD was proposed as NADP-dependent alcohol dehydrogenase with a weak affinity toward short- and medium- chain alcohols [7]. Later studies characterized this enzyme as a NADPH-dependent aldehyde reductase with a broad range of substrates, such as 3-hydroxypropionaldehyde [8], propanaldehyde, isobutylaldehyde [3], acrolein, and malondialdehyde [2,9]. The enzyme's biological role was also evident in the reduction of various reactive aldehydes derived from membrane lipid peroxidation [9] and mitigation of furfural toxicity [10–12]. Other biosynthetic efforts with YqhD have involved its utilization in the production of aromatic alcohols [13], ethylene glycol, 1-butanol [14], 1,4-butanediol, acetol [15,16], 1,2-propanediol [17] and, in a pathway using CO2, 1-butanol [18].

**Citation:** Verma, R.; Ellis, J.M.; Mitchell-Koch, K.R. Dynamic Preference for NADP/H Cofactor Binding/Release in *E. coli* YqhD Oxidoreductase. *Molecules* **2021**, *26*, 270. https://doi.org/10.3390/ molecules26020270

Academic Editors: Marilisa Leone Received: 29 November 2020 Accepted: 4 January 2021 Published: 7 January 2021

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Just a few years after YqhD's characterization, it became a candidate for protein engineering studies toward the biological production of useful chemicals and fuels, including valorization of biomass-derived precursors. Protein engineering approaches have produced variants of YqhD with improved affinity and higher catalytic efficiency toward 3 hydroxypicolinic acid for the production of 1,3-propanediol [19]. Engineered strains for the production of 1,3-propanediol from D-glucose [20] and glycerol [21,22] have also utilized YqhD. The broad enzyme substrate scope, combined with this report on the dependence of enzyme conformational dynamics on cofactor oxidation state, makes YqhD useful as a model system to better understand NADP-dependent zinc bound oxidoreductases [23–26]. Despite the abundance of structural and kinetics data for the YqhD oxidoreductase, there are no reports (experimental or simulations) until now on the structure and dynamics of this enzyme in solution.

Herein, we report results from a set of molecular dynamics (MD) simulations of YqhD enzyme with both oxidized and reduced cofactors (NADP and NADPH) in aqueous solution. The study aims to rationalize the enzyme preference toward NADPH over NADP as a cofactor. The crystal structure [7] (PDB ID: 1OJ7) is the starting point for our simulations. In the crystal structure of the YqhD holoenzyme, the bound NADP cofactor is modified to NADPH(OH)<sup>2</sup> due to oxidative stress during the crystallization process [7]. Figure 1 shows the dimeric structure of YqhD, which crystallizes as a tetramer in the asymmetric unit, as well as geometries of the modified and native cofactors (NADPH(OH)2, NADP, and NADPH). YqhD shows a lower Michaelis constant for NADPH [11] (K<sup>M</sup> = 0.008 mM) than NADP [27] (K<sup>M</sup> = 0.15 mM) and, concomitant with cofactor reactivity, a lower Michaelis constant for 1-butyaldehyde [9] (K<sup>M</sup> = 0.67 mM) compared to 1-butanol [7] (K<sup>M</sup> = 36 mM). Our simulation work of the YqhD homodimer has found that the cofactor oxidation state has a profound effect on enzyme structure and dynamics, which is consistent with differences in enzyme efficacy toward alcohols versus aldehydes. **2021**, , x FOR PEER REVIEW 3 of 17

α **Figure 1.** (**a**) YqhD dimer (comprised of monomers A and D) is in cartoon representation with the α-helical and Rossmanntype domains colored in blue and pink, respectively. The shaded region shows the cofactor-binding site of monomer D. Zn2+ is located at the interdomain face within the cofactor-binding site and is shown in orange colored VDW representation. The NADP cofactor is in licorice representation colored by elements (nitrogen, oxygen, hydrogen, carbon and, phosphorus in blue, red, white, carbon, and tan color, respectively). (**b**) NADP cofactor is present in the crystal structure as NADPH(OH)<sup>2</sup> (with modification at C5 and C6 position in the nicotinamide moiety colored by elements and N and O atomic labeling) and in the MD simulations as NADPH and NADP in blue and red color, respectively. The black ovals highlight the nicotinamide moiety.

It has been observed previously that differences in cofactor oxidation states induce protein conformational changes [25,28] and also influence the cofactor binding affinities [29]. Along these lines, structural differences in cofactor binding for FucO oxidoreductase, a NADH-dependent enzyme with similarities to YqhD, were recently reported [30], while

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α

conformational changes in cofactor were shown to be critical in the pathway toward the transition state for horse liver alcohol dehydrogenase [31]. Recent work on product release, a rate-limiting step in dihydrofolate reductase (DHFR), indicates that NADPH induces product release through steric repulsion during conformational sampling of the closed excited state [32]. There is some variation in conformational dynamics (rates between exchange of states) when the DHFR-product complex is bound to oxidized (NADP) vs. reduced (NADPH) cofactor, but conformational exchange rates measured by nmR are on the same order of magnitude (1890 ± 80 s−<sup>1</sup> bound to NADPH vs. 1420 ± 70 s−<sup>1</sup> when bound to NADP<sup>+</sup> ).

The present work has gone beyond structural effects to computationally study how dynamics are affected by cofactor oxidation/reduction. To gain insight into dynamical preference of the YqhD protein towards NADPH over NADP cofactor, the article is organized as follows: the details of MD simulations are reported in the Methods section; the results section has the analysis of MD trajectories, with the main focus on structural and dynamical properties of YqhD enzyme and NADP/H cofactor binding; next, we discuss our results, focusing on the effect of cofactor oxidation/reduction on structure-dynamics and activity of the enzyme.
