Escherichia coli alcohol dehydrogenase YahK is a protein that binds both iron and zinc

Protein purification

The alcohol dehydrogenases YahK and AdhP from E. coli are zinc-dependent medium-chain ADHs, consisting of 349 and 336 amino acids, respectively (Pick et al., 2013; Thomas et al., 2013). Both proteins exhibit a zinc-binding GHEX₂GX₅ (G/A)X₂ (I/V/A/C/S) protein motif, as well as the GX_1–3GX_1–3G pattern located in the nucleotide-binding region (Pick et al., 2013; Thomas et al., 2013). Protein alignment analysis revealed a significant similarity of 27% with an e-value of 2e⁻²⁹ between YahK and AdhP. Therefore to facilitate the study of E. coli alcohol dehydrogenases YahK metal-binding properties and activity, the well-studied AdhP was used as a reference in this study. The wild-type E. coli genomic DNA was utilized as the template, while plasmid pET28b⁺ (Novagen, Darmstadt, Germany) served as the expression vector. The YahK and AdhP genes were amplified through polymerase chain reaction using primers containing oligonucleotides with HindIII and NcoI restriction endonuclease site (Table 1). Recombinant YahK and AdhP were subsequently expressed in the E. coli BL21 (DE3) strain, cultivated in LB medium.

Purification of the proteins with six histidines at the C-terminus were purified following the previously described protocol (Tan et al., 2017). Harvested cells were resuspended in protein preservation solution (buffer A: 500 mM NaCl, 20 mM Tris-HCl, pH 8.0). The histidine-rich recombinant proteins were then purified using a nickel-ion metal-chelated affinity chromatography medium, Ni-NTA. Buffer A served as the running buffer, while buffer B (500 mM NaCl, 20 mM Tris-HCl, 500 mM imidazole, pH 8.0) functioned as the elution buffer. A second purification step involved size exclusion chromatography (SEC) utilizing a Superdex 200 16/60 gel filtration column from General Electric Healthcare, which was equilibrated with buffer A, excluding imidazole. Protein purity was verified by SDS-PAGE and protein concentration was determined employing a U3900 (Hitachi High-Tech Co., Tokyo, Japan) instrument, utilizing the published theoretical extinction coefficients. The protein concentration was also determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA), employing bovine serum albumin as the standard. Notably, both methods yielded highly comparable results.

When evaluating the metal binding properties of YahK, M9 minimal medium with clearly defined components was chosen to induce expression (CSHL, 2010). Recombinant YahK was expressed in E. coli cells grown in M9 minimal medium supplemented with a fixed concentration of ferric citrate (50 µM), glycerol (0.2%), thiamine (5 µg/ml), and 20 amino acids (100 µg/ml) at 37 °C. Different concentrations of zinc sulfate (0 to 8 μM) were added to the M9 minimal medium before protein expression was induced by L-Arab. The absorbance peak amplitude at 330 nm for purified YahK was utilized to assess the extent of iron binding within YahK.

Alcohol dehydrogenase activity assay

The alcohol dehydrogenase activity assay was conducted according to the protocol described by Pick et al. (2013). Alcohol dehydrogenase utilizes NADP⁺ as a coenzyme to catalyze the dehydrogenation of alcohol into acetaldehyde. Simultaneously, NADPH is produced, which exhibits a characteristic absorption peak at 340 nm. The enzymatic activity assay reaction mixture consisted of 20 mM ethanol, 2 mM NADP⁺, 100 mM N,N-Bis(2-hydroxyethyl)glycine (Bicine) at pH 8.5, and 50 mM phosphate buffer at pH 7.4. When measuring ADH enzyme activity, the reaction system for enzyme activity measurement should be thoroughly mixed. Subsequently, 170 µL of the reaction solution was transferred to a cuvette and incubated at 37 °C for 2 min. Then, 30 µL of purified protein was added to the system. It was mixed by inversion, and the monitoring of the change in OD₃₄₀ was initiated immediately. The change in OD₃₄₀ reflects the activity of alcohol dehydrogenase. The unit of enzyme activity was defined as the quantity of NADPH generated by 1 µM of enzyme during 1 min of enzymatic activity.

Metal content analysis

The determination of total iron content in protein samples was carried out using the iron indicator FerroZine, following the procedure described earlier (Cowart, Singleton & Hind, 1993). Briefly, protein samples were heated to 85 °C for 30 min with 500 µM ferroZine and 2 mM L-cysteine. After incubation, the protein samples were centrifuged for 5 min at 12,000 rpm to remove the precipitate. The concentration of the iron-ferroZine complex was measured at 564 nm using an extinction coefficient of 27.9 mM⁻¹ cm⁻¹. For the assessment of total zinc content in protein samples, the zinc indicator PAR (4-(2-pyridylazo)-resorcinol) method was employed (Bae et al., 2004). To the protein solution under investigation, add 2 mM DTT and 2 mM PAR followed by the addition of hydrogen peroxide with a final concentration of 10 mM. Allow the mixture to incubate at room temperature for 20 min, then centrifuge it at 12,000 rpm for 5 min to remove denatured protein precipitate and collect the supernatant. Measure the absorbance at OD_500nm using a UV-visible spectrophotometer and determine the zinc concentration in the solution based on an absorption coefficient of 66.0 mM⁻¹ cm⁻¹. The quantification of zinc and iron in the purified proteins was accomplished through inductively coupled plasma emission spectrometry (ICP-MS). It is noteworthy that both metal content analyses yielded congruent results.

Site-directed mutagenesis

The plasmid YahK-pET28b⁺ was previously constructed as described earlier. Based on information obtained from the Ecogene and NCBI databases, it was ascertained that YahK can bind two zinc ions: active zinc binding sites Cys40, His62, and Cys158, as well as structural zinc binding sites Cys93, Cys96, Cys99, and Cys107. Mutant strains involving alterations at the Cys40 and Cys99 sites, where the original amino acids were substituted with alanine, were generated using Mut Express MultiS Fast Mutagenesis Kit (Vazyme Biotech Co., Ltd. Nanjing, China). The PCR primers used in this endeavor are detailed in Table 1, and the specific mutations were corroborated through direct sequencing.

Statistical analysis

The data analysis was performed using KaleidaGraph (Synergy Software, Inc., Eden Prarie, MN, USA) and GraphPad Prism version 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). Data were expressed as the mean ± standard deviation (mean ± SD) from three independent experiments, and differences between groups were analyzed using an unpaired t-test or one-way ANOVA followed by a Tukey test. P < 0.05 was considered statistically significant.

E. coli alcohol dehydrogenase YahK has an iron-binding affinity

To study the metal-binding properties of YahK, we expressed YahK and AdhP in BL21 (DE3) cells cultured in LB medium under aerobic growth conditions due to their shared zinc-binding domain (Pick et al., 2013; Thomas et al., 2013). By conducting UV-Vis absorption measurements, a significant absorption peak at 330 nm was observed for YahK compared to AdhP (Fig. 1A), indicating its potential iron-binding capability. Regarding metal content analysis, it was found that each monomeric unit of YahK contained an average of 0.26 ± 0.01 iron atoms and 1.4 ± 0.06 zinc atoms (Figs. 1B and 1C). Furthermore, the iron binding ratio of YahK was significantly higher than that of AdhP, while the zinc binding ratio remained relatively constant. These preliminary observations suggested that E. coli YahK was capable of binding to both iron and zinc. Additionally, when compared with the control group’s AdhP protein, YahK exhibited significantly higher ADH activity (Fig. 1D).

In conclusion, YahK demonstrated a strong affinity to bind both iron and zinc in vivo, with its elevated iron binding ratio showing a positive correlation with increased catalytic activity. Overall, substantial alterations in iron binding may play a pivotal role in enhancing YahK’s catalytic activity. Figure 1E depicts the enzymatic reaction mechanism.

Iron and zinc binding activities of E. coli YahK in M9 minimal medium

To directly assess the metal-binding characteristics of proteins and exercise better control over metal content in the growth medium, we opted for M9 minimal medium to induce the expression of the target protein. As depicted in Fig. 2A, purification of YahK from E. coli cells cultured in M9 minimal medium under aerobic conditions revealed a prominent absorption peak at 330 nm. Furthermore, analysis of total metal content demonstrated that each YahK monomer contained an average of 0.40 ± 0.02 iron atoms and 0.25 ± 0.04 zinc atoms (Fig. 2B). In parallel experiments, supplementation with additional iron ions (50 µM) to the M9 minimal medium significantly intensified the absorbance peak of YahK at 330 nm, resulting in each YahK monomer contained 0.60 ± 0.02 iron atoms and 0.15 ± 0.04 zinc atoms (Figs. 2A and B). Conversely, the introduction of exogenous zinc (50 µM) into the M9 minimal medium resulted in YahK monomers containing only trace amounts of iron (0.02 ± 0.01 atoms), while being enriched with zinc (2.0 ± 0.04 atoms) (Figs. 2A and B).

We further investigated the effect of different culture conditions on the activity of YahK by using NADP⁺ as its specific substrate. Experimental findings demonstrate that in the M9 minimal medium supplemented with or without 50 µM ferric citrate, the rate of NADPH generation was relatively low. Figure 2C illustrated a significant increase in the rate of NADPH generation upon addition of zinc sulfate (50 µM), indicating that zinc presence substantially enhances YahK’s catalytic efficiency. In summary, our results reveal pronounced iron and zinc binding activity exhibited by YahK in the M9 minimal medium. Specifically, we observed heightened affinity between YahK and iron in its presence, while also noting increased zinc binding affinity alongside notable NADPH generation activity when zinc is present. This result further highlights the apparent correlation between the metal binding capacity of YahK and the specific metal content in the medium.

Regulation of YahK activity by iron and zinc binding in E. coli

To investigate the regulatory role of metal iron and zinc in the activity of YahK within cells, we induced the expression of YahK in the M9 minimal medium containing a fixed iron concentration (50 µM) and gradually increased zinc concentration (0–8.0 µM) under aerobic conditions. In the experiment, as the exogenous zinc concentration in the M9 minimal medium increased, a significant change was observed in the absorption peak at 330 nm. Specifically, as the zinc sulfate concentration increased from 0 to 2.4 µM, there was a sharp rise in the number of zinc atoms per protein monomer while the number of iron atoms gradually decreased and approached zero with increasing zinc concentration. These findings suggested a preference for zinc binding over iron by YahK when both metals were present. Concomitantly, upon raising zinc sulfate concentration from 0 to 2.4 µM, YahK activity exhibited rapid enhancement reaching an apparent plateau, indicating substantial activation of YahK in the presence of adequate zinc (Figs. 3A–3C). In vitro experiments also support this idea (Fig. 3D). Additionally, in E. coli cells grown in the M9 minimal medium with a fixed zinc concentration (1 µM), we attempted to use excess iron to compete with zinc for binding to YahK. However, even when the iron concentration gradient in the medium was increased to 100 µM, we were unable to obtain completely metal-free Fe-YahK, and the results of the in vitro experiment were consistent with this (Fig. S1). Taken together, the results suggested that the metal-binding properties and catalytic activity of YahK are significantly influenced by the specific metal content in the culture medium.

Localization of the iron-binding site of YahK

To elucidate the iron-binding sites of YahK, we constructed YahK mutants in which the active zinc-binding site Cys-40 and the structural zinc-binding site Cys-99 were replaced with serine (S). Wild-type YahK and its mutants were then expressed in E. coli cells grown in M9 minimal medium supplemented with ZnSO₄ or ferric citrate (50 µM), followed by purification of each protein from the E. coli cells. As illustrated in Figs. 4A–4C, the YahK-C40S mutant expressed in M9 minimal medium supplemented with 50 µM ferric citrate exhibited a lower absorption peak at 330 nm compared to the wild-type YahK, yet still maintained a distinct peak. The iron content in YahK-C40S was comparable to that of wild-type YahK. In contrast, the YahK-C99S mutant showed minimal absorption at the 330 nm peak, demonstrating significantly reduced iron affinity. Both C40S and C99S mutations markedly decreased the enzymatic activity of YahK.

Additionally, when 50 µM ZnSO₄ was added to the M9 minimal medium, zinc content in the YahK-C40S mutant was observed to be only half of that in wild-type YahK, whereas the zinc content in YahK-C99S nearly matched the levels found in wild-type YahK. The alcohol dehydrogenase activity of both mutants, YahK-C40S and YahK-C99S, was almost completely abrogated (Figs. 4D–4F).

The ablation of the active zinc-binding site Cys-40 predominantly affects zinc binding and the corresponding biological activity, while exerting minimal impact on iron binding. Conversely, mutation of the structural zinc-binding site Cys-99 substantially impairs YahK’s iron-binding capacity and enzymatic activity. These findings illustrate that the Cys-99 residue is critical for iron binding and is involved in regulating both iron and zinc binding. Collectively, these experiments underscore the essential roles of Cys-40 and Cys-99 residues in the functional mediation of YahK.

Iron maintains the stability of YahK

We expressed YahK in E. coli cells cultured in M9 minimal medium supplemented with either 50 µM ferric citrate or 50 µM zinc sulfate. Subsequently, the bacterial cells were lysed (with consistent turbidity) using sonication followed by centrifugation to separate the soluble proteins (supernatant) from the insoluble proteins (sediment, potentially containing inclusion bodies). Whole-cell protein electrophoresis analysis was then performed, and the gel was stained with Coomassie Brilliant Blue. As depicted in Fig. 5, compared to YahK induced for expression in zinc sulfate-supplemented medium, a significantly higher amount of YahK was observed in the sediment when cultured in both iron citrate-supplemented medium and metal-free M9 minimal medium, suggesting a propensity of YahK to form inclusion bodies during overexpression under these conditions.