123rd General Meeting of the KCS

Type Poster Presentation
Area Life Chemistry
Room No. Exhibition Hall 2
Time 4월 19일 (금요일) 11:00~12:30
Code LIFE.P-362
Subject Structural basis for low catalytic activities in the two minor beta-carbonic anhydrases from the filamentous fungi Aspergillus fumigatus
Authors Najin Kim, Songwon Kim1, Mi Sun Jin2,*
Life Science, Gwangju Institute of Science and Technology, Korea
1School of Life Sciences, Gwangju Institute of Science and Technology, Korea
2Division of Life Science, Gwangju Institute of Science and Technology, Korea
Abstract In the fungal kingdom, the beta-carbonic anhydrases (beta-CAs) are widely distributed zinc-metalloenzymes that play essential roles in growth, survival, development and virulence. In particular, the majority of filamentous ascomycetes possess multiple beta-CA isoforms in which the majors and minors have been characterized. Herein, we tested for in vitro catalytic behaviors of the two minor beta-CAs, CafC and CafD, from Aspergillus fumigatus, and confirmed that both enzymes exhibit low CO2 hydration activities. To understand the structural basis of their low activities, we further performed X-ray crystallographic and site-directed mutagenesis studies. Both enzymes exist as homodimer. Similar to other Type-I beta-CAs, CafC active site reveals the “open” conformation in which the zinc ion is tetrahedrally coordinated by three residues (C36, H88 and C91) and a water molecule. However, L25 and L78 on the rim of the catalytic entry site protrude into the active site cleft, partially occluding access to it. Consistent with our structural analysis, single (L25G or L78G) and double mutants provide functional evidences that widening the entrance to the active site greatly accelerates the catalytic reaction. By contrast, CafD shows a typical Type-II “closed” conformation in which a zinc-bound water is replaced by aspartic acid (D36). The most likely explanation of this result is that a completely conserved arginine is substituted to glycine (G38), therefore, D36 is not allowed to undergo a conformational change by forming a D-R pair that can leave a space for a zinc-bound water and switch the enzyme to be active. Furthermore, CafD structure reveals the presence of a copper ion in the interface, which may contribute to stabilize the dimeric assembly.
E-mail nhjynhss@gist.ac.kr