Structure and activity of N-terminal truncated versions of Arabidopsis proline dehydrogenase isoforms

FABRO G; CISLAGHI A; CONDAT F; ALVAREZ ME

Paraná

Congreso; LIV Reunión Anual de SAIB; 2018

Proline dehydrogenase (ProDH) is a mitochondrial enzyme that catalyses the oxidation of proline (Pro) to Δ1-pyrroline-5-carboxylate (P5C).Arabidopsis contains two active ProDH isoforms AtProDH1 and AtProDH2.Modelling of A. thaliana ProDH isoforms over the structure of E.coli ProDH showed that these proteins adopt the shape of a distorted (ȕα)13 TIM-barrel fold. The γD structure begins with γ α-helixes located ontop of the barrel simulating a ―cap‖. Below this cap is buried the last α-helix of the protein carrying conserved active site aminoacids contacting Pro and the cofactor FAD. The N-terminal primary sequence of ProDHs from all kindoms of life is poorly conserved. To investigate thefunctional impact of this ―cap‖ in catalysis and oligomerization of ProDH, we constructed deletion variants of AtProDHs. Both protein isoformswere expressed as recombinants in E. coli, using MBP as affinity purification tag. Proteins were tested for their activity in vitro, revealing thatthe ―cap‖ is essential for ProDH activity. Oligomerization status was addressed by non-denaturing gel electrophoresis. Results of these assayswill be discussed.