Identification and characterization of catalytically important residues in the active site of human GDP-fucose synthase

master's thesis

• Title (croatian): Identifikacija i karakterizacija važnih aminokiselina u aktivnom mjestu humane GDP-fukoza sintaze
• Author: Klara Kastner
• Mentor: Anita Slavica (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Biochemical Engineering); (Laboratory for Biochemical Engineering, Industrial Microbiology and Malting and Brewing Technology); Zagreb
• Defense date and country: 2022-01-28, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Biotechnology

Abstract

Human GDP-fucose synthase (hGFS) catalyzes three distinct reactions within a single active site: two epimerizations and one reduction. Wild-type hGFS and 11 targeted mutants (C116S, C116A, C116D, H189N, H189D, Y143F, K149A, S114A, N79A, N140A, K282A) were designed for identification and characterization of important residues. Mutants C116A, C116D and H189D do not possess activity towards the substrate, which indicates importance of those residues in the epimerization reactions. Based on the obtained kinetic parameters, N140A mutant seems to be involved in binding and positioning of the substrate. Km value of K282A mutant (482.3 mmol Lˉ¹) indicates that this residue is possibly employed in the substrate binding. Mutants C116S and N140A generate additional product (presumably GDP-Alt). Reaction sequence in the active site of the hGFSs was assessed by following production of deuterated compounds (GDP-4k6d-Man and GDP-Fuc). First epimerization reaction occurs at C-3”, the second one at C-5”, and subsequently the reduction at C-4”.

Abstract (english)

Humana GDP-fukoza sintaza (hGFS) katalizira tri različite reakcije u jednom aktivnom mjestu: dvije epimerizacije i jednu redukciju. Divlji tip hGFS i 11 ciljanih mutanata (C116S, C116A, C116D, H189N, H189D, Y143F, K149A, S114A, N79A, N140A, K282A) je dizajnirano za identifikaciju i karakterizaciju važnih aminokiselina. Mutanti C116A, C116D i H189D ne iskazuju aktivnost prema supstratu, što bi moglo ukazivati na važnost ovih aminokiselina u reakcijama epimerizacija. Na temelju dobivenih kinetičkih parametara može se zaključiti da je mutant N140A uključen u vezanje i pozicioniranje supstrata u aktivnom mjestu. Km vrijednost mutanta K282A (482.3 mmol Lˉ¹) ukazuje da je ova aminokiselina možda uključena u vezanje supstrata u aktivno mjesto enzima. Mutanti C116S i N140A proizvode dodatni produkt (pretpostavlja se GDP-Alt). Redoslijed reakcija u aktivnom mjestu svih hGFS predložen je na temelju nastanka deuteriranih spojeva (GDP-4k6d-Man i GDP-Fuc). Prva epimerizacija odvija se na C-3”, druga epimerizacija na C-5”, a slijedeća je reakcija redukcije na C-4”.

• Keywords: GDP-fucose synthase; NDP-sugars; SDR-enzymes
• Keywords (english): GDP-fukoza sintaza; NDP-šećeri; SDR-enzimi
• Language: english
• URN:NBN: urn:nbn:hr:159:527952
• Study programme: Title: Bioprocess Engineering; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva (magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Closed access
• Created on: 2022-05-26 12:51:04