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. 2018 Oct 31;13(10):e0206260.
doi: 10.1371/journal.pone.0206260. eCollection 2018.

Crystal Structure and Functional Characterization of a Cold-Active Acetyl Xylan Esterase (PbAcE) From Psychrophilic Soil Microbe Paenibacillus Sp

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Free PMC article

Crystal Structure and Functional Characterization of a Cold-Active Acetyl Xylan Esterase (PbAcE) From Psychrophilic Soil Microbe Paenibacillus Sp

Sun-Ha Park et al. PLoS One. .
Free PMC article

Abstract

Cold-active acetyl xylan esterases allow for reduced bioreactor heating costs in bioenergy production. Here, we isolated and characterized a cold-active acetyl xylan esterase (PbAcE) from the psychrophilic soil microbe Paenibacillus sp. R4. The enzyme hydrolyzes glucose penta-acetate and xylan acetate, reversibly producing acetyl xylan from xylan, and it shows higher activity at 4°C than at 25°C. We solved the crystal structure of PbAcE at 2.1-Å resolution to investigate its active site and the reason for its low-temperature activity. Structural analysis showed that PbAcE forms a hexamer with a central substrate binding tunnel, and the inter-subunit interactions are relatively weak compared with those of its mesophilic and thermophilic homologs. PbAcE also has a shorter loop and different residue composition in the β4-α3 and β5-α4 regions near the substrate binding site. Flexible subunit movements and different active site loop conformations may enable the strong low-temperature activity and broad substrate specificity of PbAcE. In addition, PbAcE was found to have strong activity against antibiotic compound substrates, such as cefotaxime and 7-amino cephalosporanic acid (7-ACA). In conclusion, the PbAcE structure and our biochemical results provide the first example of a cold-active acetyl xylan esterase and a starting template for structure-based protein engineering.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Crystal structure of PbAcE and multiple sequence alignment.
(A) Overall structure of PbAcE is shown in front and 90° rotated views. Ribbon representation of PbAcE, with the β-strands in forest green and α-helices in red. The conserved catalytic triad residues are shown as grey stick models. (B) Sequence alignment of PbAcE with secondary structure. Aligned sequences include PbAcE, TmAcE (UniProtKB id: Q9WXT2), BsAcE (UniProtKB id: P94388), and BpAcE (UniProtKB id: Q9K5F2). The β5–α4 loop region (residues 119–152), called the β-interface, is boxed in sky blue. The Tyr133 and Leu144 residues located in β-interface region are indicated above the alignment residues with a black rectangle and triangle, respectively. The catalytic triad residues of Ser185, Asp274, and His303 are indicated with black circles.
Fig 2
Fig 2. Active site and β-interface of PbAcE.
(A) Active site and substrate binding site are circled in salmon and purple, respectively. Side chains of catalytic triad and residues located at substrate binding site are indicated by stick models (forest green). The β-interface region is represented in marine. (B) PbAcE forms a donut-shaped hexamer containing a trimer of dimers. (C) The dimer interface between each pair of monomers contains the β-interface region (marine). Close-up view of β-interfaces of PbAcE (D), BsAcE (E), and TmAcE (F) depicted in forest green, yellow, and orange, respectively. Specific residues that affect the conformation of the β-interface are shown as stick models. Hydrogen bonds in the β-interface are represented as red dashed lines.
Fig 3
Fig 3. Comparison of different entrance conformations.
(A) The β4–α3 and β5–α4 loop (β-interface) regions form a substrate gate in PbAcE (forest green). (B) Superposition with BsAcE (yellow) shows the difference in the β4–α3 loop region. (C) Superposition with TmAcE (orange) shows the difference in the β5–α4 loop (β-interface) region. A bound OIA molecule is shown as a cyan stick model. Surfaces of PbAcE (D), BsAcE (E), and TmAcE (F) represent the entrances for substrates, circled with red dashed lines. Only the β4–α3 and the β5–α4 loop regions are colored as above the figure, while the remaining protein is in gray.
Fig 4
Fig 4. Substrate specificity of PbAcE.
(A) A pH shift assay was performed to measure the hydrolytic activity of acetylated carbohydrate substrates. The hydrolytic activities toward (B) lipids and (C) tertiary alcohol esters were also examined under the indicated reaction times. (D) The hydrolysis of antibiotic-related compounds by PbAcE wild-type and S185A inactive mutant: 7-ACA, 7-aminocephalosporanic acid; CPC, cephalosporin C. Acetic acid released in the enzyme reaction changed the solution color from red to yellow.
Fig 5
Fig 5. Hydrolytic activities toward pNP and naphthyl esters.
The relative enzyme activity of PbAcE for p-nitrophenyl (pNP) esters with varying acyl chain lengths from C2 to C8 (A) and α-,β-naphthyl ester derivatives (B). The change in the initial rate of the reaction at different concentrations of (C) pNP-acetate and (D) pNP-butyrate are shown. (E) Relative activities and kinetic parameters of PbAcE towards these two substrates were determined from the initial rate measurements. The highest activity obtained was set as 100%. All measurements were performed in triplicate.
Fig 6
Fig 6. Effects of temperature and organic solvents on the activity of PbAcE.
(A) Enzyme activity was measured at various temperatures. (B) Thermal stability was determined by assaying residual enzyme activity after incubation of PbAcE for different time periods at the temperatures indicated. (C) After incubation of PbAcE wild-type and L144R mutant at 70°C, residual activities were measured. (D) Chemical stability of PbAcE was investigated after exposure to various organic solvents for 1 h and determination of residual activities, expressed relative to the original activity. All measurements were performed in triplicate using pNP-C2 as a substrate.
Fig 7
Fig 7. Immobilization of PbAcE.
(A) The relative activities of PbAcE CLEAs cross-linked by different concentrations of glutaraldehyde. (B) Reusability of PbAcE CLEAs was compared to that of the soluble enzyme for 18 cycles. (C) Acetylation activity of PbAcE CLEAs on xylan was observed via gas chromatography. (D) The relative activities of PbAcE CLEAs and PbAcE mCLEAs were compared to that of the soluble enzyme. The PbAcE mCLEAs were prepared with different amounts of PbAcE and 500 μg of MNPs. The activity of soluble PbAcE was set as 100%. All measurements were performed in triplicate using pNP-C2 as a substrate.

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Grant support

This work was supported by the Korea Polar Research Institute [grant number PE18210]; the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (MSIP) [application study on Arctic cold-active enzymes degrading organic carbon compounds; NRF grant number NRF-2017M1A5A1013568]; and the Korea Polar Research Institute (KOPRI) [grant number PN18082].
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