123rd General Meeting of the KCS

Type Oral Presentation
Area Oral Presentation of Young Life Chemist
Room No. Room 303
Time THU 09:30-09:45
Code LIFE.O-3
Subject Structural basis for low catalytic activities in the two minor β-carbonic anhydrases from the filamentous fungi Aspergillus fumigatus
Authors Songwon Kim, Mi Sun Jin1,*
School of Life Sciences, Gwangju Institute of Science and Technology, Korea
1Division of Life Science, Gwangju Institute of Science and Technology, Korea
Abstract In the fungal kingdom, the β-carbonic anhydrases (β-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 β-CA isoforms in which the majors and minors have been characterized. Herein, we tested for in vitro catalytic behaviors of the two minor β-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 β-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 within the β-CA family 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 songwon@gist.ac.kr