W-motif exchange between beta-propeller proteins.
The similarity between the structural scaffold of PQQGDH and that of sialidase in the absence of any similarity in the primary structure, catalytic function and substrate recognition encouraged us to attempt a W-motif exchange between these enzymes. By substituting one W-motif in PQQGDH with one from sialidase, a chimeric PQQGDH was constructed, and its enzymatic properties were investigated. The overexpression of the chimeric enzyme resulted in the formation of an inclusion body. However, the refolding procedure resulted in a soluble chimeric enzyme with PQQGDH activity showing similar secondary-structure components as native PQQGDH. In contrast to native PQQGDH, the chimeric PQQGDH showed thermal instability and sensitivity to EDTA; this difference might have been due to the incomplete compatibility of the inserted W-motif. The potential of W-motif replacement was also discussed in view of the possible molecular evolution/engineering of beta-propeller structures.
Tachino
Atsushi
A
Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
Igarashi
Satoshi
S
Sode
Koji
K
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D005948:Glucose Dehydrogenases / Q000737:chemistry*; D008954:Models, Biological; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000737:chemistry*; D015202:Protein Engineering; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D011993:Recombinant Fusion Proteins; D005948:Glucose Dehydrogenases; C033955:glucose dehydrogenase (pyrroloquinoline-quinone); D009439:Neuraminidase