大家好，今天为大家带来的文章是2024年2月16发表在ACS Catalysis上的“Bacterial Lactonases ZenA with Noncanonical Structural Features Hydrolyze the Mycotoxin Zearalenone”，通讯作者是帝斯曼-芬美意动物营养与健康研发中心的Wulf-Dieter Moll和布尔诺圣安妮大学医院国际临床研究中心的Zbynek Prokop。
Zearalenone (ZEN) is a fungal estrogen, polyketone, produced by Fusarium oryzae and other plant-pathogenic Fusarium species. Contamination of ZEN in cereals is common, and its hydrolysis and detoxification using fungal lactones has been explored. Here, the authors report the isolation of a ZEN hydrolytic active Rhodococcus erythropolis PFA D8−1, cloning the gene encoding the α/β hydrolase ZenA on the linear plasmid pSFRL1, and biochemical identification of its 9 homologs. In addition, the authors report on R. Brown, a phyto-site oriented mutagenesis and structural analysis. dimer of erythropolis and tetramer of the more heat-resistant Streptomyces coelicoflavus (without binding ligands). The X-ray crystal structure reveals not only the typical features of α/β hydrolases, including the Ser-His-Asp catalytic ternary domain, but also the unusual characteristics of α/β hydrolases, including the uncommon oxyanion hole motif and peripheral antiparallel short β sheets involved in tetrameric interactions. Pre-steady-state kinetic analysis of ZenARe and ZenAScfl determined an equilibrium rate-limiting step of the reaction cycle, which can vary with temperature. Some new bacterial ZEN lactases have lower KM and higher kcat than known fungal ZEN lactases, which may contribute to the development of enzyme technology to degrade ZEN in feed or food.
- 1. Cloning of the ZEN lactesterase gene
The authors enriched several bacterial mixed cultures extracted from the soil, and the results showed ZEN degradation. Subsequently, the authors streaked the culture to obtain single colonies, and the culture of each colony was tested for ZEN transformation activity. The isolated PFA D8−1 showed rapid and robust ZEN transformation activity, and 16S rDNA sequencing identified it as R. erythropolis。
Previous studies have shown that HZEN is the primary metabolite of PFA D8−1 conversion to ZEN. R. erythropolis DSM 43066T and R. erythropolis DSM 43066T were determined. erythropolis PR482 has no hydrolytic activity against ZEN. Rhodospora PFA D8−1 lysate also converts ZEN to HZEN, but biomass needs to be exposed to ZEN prior to lysis, and the activity of the uninduced biomass lysate is low.
PFA D8−1 of Rhospora was sequenced whole-genome and 9 sequencing scaffolds covering 7.08 Mb were obtained (sequences were deposited in DDBJ/ENA/GenBank as BioProject PRJNA884193). Seven of the scaffolds were well matched to the Rhospora PR4 genome, but scaffolds 1 and 8 (covering a total of 659 kbp) did not show a match. Pulsed-field gradient gel electrophoresis of Rhodospora PFA D8−1 DNA showed a band at 660 kbp.
Linear megaplasmids are typical of Rhodosporum and often contain genes for the catabolism of exogenous drugs. The authors named the new megaplasmid pSFRL1 and speculated whether there was a gene capable of degrading ZEN. The authors attempted to isolate pSFRL1 DNA from pulsed-field electrophoresis gels and cloned the partially digested pSFRL1 DNA into the E. coli-Rhodococcus shuttle vector pMVS301.85. However, the cumbersome preparation of pSFRL1 DNA means that genome-wide DNA libraries are more suitable for cloning and functional screening of libraries prepared from isolated pSFRL1 DNA.
The Rhodospora PFA D8−1 genomic library was cloned by isolating genomic DNA, partially digesting it with a restriction enzyme (HinIII) with a 4 base pair recognition site, and ligating the fragments into the plasmid vector pMVS301. A peg-mediated protoplast transformation protocol was used to convert the library into Rhodospora PR4, with an average of 11.5 kbp of Rhodospora PFA D8−1 genomic DNA inserted into each clone. Clone P1G2 with 7781 bp inserted in pMVS301 was identified by screening clones with zen hydrolytic activity. This insert is localized to pSFRL1 and contains a 987 bp ORF (DDBJ/ENA/GenBank login as OAX51_31155) that is expected to encode α/β hydrolase. This ORF was amplified, inserted into pET-28a(+), transformed into E. coli BL21 (DE3) and HMS174 (DE3), and verified to have ZEN hydrolytic activity. Following the convention of bacterial gene nomenclature, the authors named the gene zenARe.
2. Enzymatic and biophysical properties of ZenARe
The c-terminal 6xhis tag on ZenARe has no effect on enzyme activity and yield in E. coli (data not shown) for the preparation of pure enzymes. ZenARe activity is highest at 38 °C (Figure 1A), at which temperature activity decreases with increasing time during incubation (Figure 1B), and within the melting temperature range determined by thermofluorescence (Figure 1C). Activity was highest at pH 8.2 at 30°C, and ZenARe remained active after incubation at pH 6.5 to pH 10 at 25°C (Figure 1D).
Figure 1
Sequence alignment can predict catalytic serine (Figure 2), conserved feature motifs present in nucleophilic bends GXSXG90 (Ser-128 in ZenARe) and catalytic histidine (His-303 in ZenARe). To identify the acidic residues of the catalytic triplet, a variant of ZenARe was prepared by site-directed mutagenesis. In ZenARe's Asp-264, in the sequence range where the acidic residues of the triplet are typically located, are possible targets. The variants D264A, D264L and D264N are catalytically active. Another conserved aspartic acid is also targeted, and none of the enzyme variants D153A, D153L, or D153N with amino acid exchange are hydrolyzed ZEN. Thus, the catalytic triplet of ZenARe is defined as Ser-128−His-303−Asp-153. In α/β hydrolases, the oxyanion residue I is usually located after the catalytic nucleophile and the oxyanion residue II is located in the ring after β-3.91. However, oxyanion residue II is usually preceded by one (GX-type) or two (GGGX-type) glycine residues, and there is no such sequence motif in the predicted sequence region of ZenARe. The absence of tyrosine residues indicates the presence of Y-type oxyanion holes.
Figure 2
- 3. Biochemical analysis of ZenARe homologs
The 14 ZenARe homologs listed in Table 1 were selected from BLAST search results and generated in E. coli using the c-terminal 6xHis-tag. Eight of these enzymes can be produced and purified in soluble form. They both catalyzed the hydrolysis of ZEN, but the measured kinetic parameters varied over a wide range (Table 1). These sequences are shown in Figure 2. The optimal temperature range is 20 ~ 50°C, and the optimal pH range is 7.0 ~ 8.5. Unfold temperature, determined by the inflection point of the thermofluorescence fluorescence intensity measured on the temperature slope, ranging from 38 °C to 61 °C. S112A, D137A, H286A and H286Y (PDB ID: 8CLP) were well expressed in E. coli, and the EEN hydrolytic activity of E. coli lysate was weak but detectable.
- Table 1
- 4.ZenARe和ZenAScfl的预稳态动力学
The authors selected ZenARe enzyme with high activity and ZenAScfl enzyme with high temperature stability for pre-homeostatic kinetic analysis, revealed the kinetic mechanism of ZEN hydrolysis by stopping flow and quenching methods, and estimated the rate and equilibrium constants associated with each catalytic step. Since the physiological temperature of 37°C is too fast, the authors used ZenARe to perform kinetic experiments at gradually decreasing temperatures, allowing them to precisely quantify each step and gain a deeper understanding of the thermodynamics of the reaction. The authors began the kinetic analysis by traditional analytical curve fitting to establish a kinetic model and obtain some preliminary estimates of the rate and equilibrium constants (Figure 3). In the second step, the results of the routine analysis fit are used as the initial parameters for the complex global numerical model (Figures 4 and 5). Overall, kinetic analysis used a complex dataset that included time-resolved information on the absolute concentrations of molecular species from quenching bursts and steady-state data, as well as time-resolved spectral data recorded using stop-flow fluorescence methods. Native tryptophan fluorescence provides a wealth of information about all the individual catalytic steps without the need for labeling. Control injections were performed with enzyme only and substrate only to verify that the observed signal could be attributed to the enzyme-substrate interaction rather than artifacts.
The contribution of individual reactive species to the fluorescence signal is first identified and quantified in the analytical data fitting (Figure 3), which is then consistently used to construct an appropriate scale function for numerical fitting. The authors describe step-by-step how to identify and quantify individual fluorescence contributions, as well as how to construct a final scaling function for simulating fluorescence kinetic data in the supporting information text "Kinetic Data Analysis and Statistics".
Figure 3
The step-by-step analysis described above by the authors made it possible to define a ZenA catalytic cycle model (Figure 4A), which includes (1) substrate binding, (2) inducing a conformational step (enzyme "closure"), (3) the first chemical step (intermediate formation), (4) the second chemical step (intermediate to product transformation), (5) the conformation of the enzyme-product complex prior to the enzyme-product complex (enzyme "opening") step, and (6) the last product release step.
Figure 4
The authors used the numerical integration of the rate equations derived from the ZenA kinetic model to model the steady-state and quasi-steady-state dynamics data globally (Figure 4A). The parameters of the analytical fit are used as the starting values for the global fit. In the global analysis, the full data set was simultaneously fitted (Figure 5) to derive the single rate and equilibrium constant for a single catalytic step as well as the activation enthalpy term (Figure 4C). Equally important, the parameters obtained by the two different methods, traditional analytical fitting and overall numerical integration, show a high degree of agreement, thus providing an important indication of the proposed kinetic model