Molecular determinants of xylose isomerase thermal stability and activity: analysis of thermozymes by site-directed mutagenesis.


Abstract

Xylose isomerases (XIs) from Thermoanaerobacterium thermosulfurigenes (TTXI) and Thermotoga neapolitana (TNXI) are 70.4% identical in their amino acid sequences and have a nearly superimposable crystal structure. Nonetheless, TNXI is much more thermostable than TTXI. Except for a few additional prolines and fewer Asn and Gln residues in TNXI, no other obvious differences in the enzyme structures can explain the differences in their stabilities. TNXI has two additional prolines in the Phe59 loop (Pro58 and Pro62). Mutations Gln58Pro, Ala62Pro and Gln58Pro/Ala62Pro in TTXI and their reverse counterpart mutations in TNXI were constructed by site-directed mutagenesis. Surprisingly, only the Gln58Pro mutation stabilized TTXI. The Ala62Pro and Gln58Pro/Ala62Pro mutations both dramatically destabilized TTXI. Analysis of the three-dimensional (3D) structures of TTXI and its Ala62Pro mutant derivative showed a close van der Waal's contact between Pro62-C(delta) and atom Lys61-C(beta) (2.92 A) thus destabilizing TTXI. All the reverse counterpart mutations destabilized TNXI thus confirming that these two prolines play important roles in TNXI's thermostability. TTXI's active site has been previously engineered to improve its catalytic efficiency toward glucose and increase its thermostability. The same mutations were introduced into TNXI, and similar trends were observed, but to different extents. Val185Thr mutation in TNXI is the most efficient mutant derivative with a 3.1-fold increase in its catalytic efficiency toward glucose. With a maximal activity at 97 degrees C of 45.4 U/mg on glucose, this TNXI mutant derivative is the most active type II XI ever reported. This 'true' glucose isomerase engineered from a native xylose isomerase has now comparable kinetic properties on glucose and xylose.

Submission Details

ID: QCN5fN8G

Submitter: Shu-Ching Ou

Submission Date: Aug. 28, 2018, 4:54 p.m.

Version: 1

Publication Details
Sriprapundh D;Vieille C;Zeikus JG,Protein Eng (2000) Molecular determinants of xylose isomerase thermal stability and activity: analysis of thermozymes by site-directed mutagenesis. PMID:10810157
Additional Information

Number of data points 196
Proteins Xylose isomerase ; Xylose isomerase
Unique complexes 14
Assays/Quantities/Protocols Experimental Assay: Heat-induced enzyme precipitation, TTXI ; Experimental Assay: Catalytic parameters (Km) of TTXI at 65°C, on glucose ; Experimental Assay: Catalytic parameters (Vmax) of TTXI at 65°C, on glucose ; Experimental Assay: Catalytic parameters (kcat) of TTXI at 65°C, on glucose ; Experimental Assay: Catalytic parameters (Km) of TTXI at 65°C, on xylose ; Experimental Assay: Catalytic parameters (Vmax) of TTXI at 65°C, on xylose ; Experimental Assay: Catalytic parameters (kcat) of TTXI at 65°C, on xylose ; Experimental Assay: Heat-induced enzyme precipitation, TNXI ; Experimental Assay: Catalytic parameters (Km) of TNXI at 80°C, on glucose ; Experimental Assay: Catalytic parameters (Vmax) of TNXI at 80°C, on glucose ; Experimental Assay: Catalytic parameters (kcat) of TNXI at 80°C, on glucose ; Experimental Assay: Catalytic parameters (Km) of TNXI at 80°C, on xylose ; Experimental Assay: Catalytic parameters (Vmax) of TNXI at 80°C, on xylose ; Experimental Assay: Catalytic parameters (kcat) of TNXI at 80°C, on xylose ; Derived Quantity: SD of Heat-induced enzyme precipitation, TTXI ; Derived Quantity: SD of Catalytic parameters (Km) of TTXI at 65°C, on glucose ; Derived Quantity: SD of Catalytic parameters (Vmax) of TTXI at 65°C, on glucose ; Derived Quantity: Catalytic parameters (kcat/Km) of TTXI at 65°C, on glucose ; Derived Quantity: SD of Catalytic parameters (Km) of TTXI at 65°C, on xylose ; Derived Quantity: SD of Catalytic parameters (Vmax) of TTXI at 65°C, on xylose ; Derived Quantity: Catalytic parameters (kcat/Km) of TTXI at 65°C, on xylose ; Derived Quantity: SD of Heat-induced enzyme precipitation, TNXI ; Derived Quantity: SD of Catalytic parameters (Km) of TNXI at 80°C, on glucose ; Derived Quantity: SD of Catalytic parameters (Vmax) of TNXI at 80°C, on glucose ; Derived Quantity: Catalytic parameters (kcat/Km) of TNXI at 80°C, on glucose ; Derived Quantity: SD of Catalytic parameters (Km) of TNXI at 80°C, on xylose ; Derived Quantity: SD of Catalytic parameters (Vmax) of TNXI at 80°C, on xylose ; Derived Quantity: Catalytic parameters (kcat/Km) of TNXI at 80°C, on xylose
Libraries TTXI and variants ; TNXI and variants
Sequence Assay Result Units