ORCID Profile
0000-0003-1742-1744
Current Organisations
University Hospitals Leuven
,
Nanyang Technological University
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Publisher: American Chemical Society (ACS)
Date: 16-11-2007
DOI: 10.1021/BI700820A
Abstract: Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) occupies a critical position in photosynthetic CO2-fixation and consequently has been the focus of intense study. Crystal-structure-guided site-directed mutagenesis studies have met with limited success in engineering kinetic improvements to Rubisco, highlighting our inadequate understanding of structural constraints at the atomic level that dictate the enzyme's catalytic chemistry. Bioselection provides an alternative random mutagenic approach that is useful for identifying and elucidating imperceptible structure-function relationships. Using the dimeric Form II Rubisco from Rhodospirillum rubrum, its gene (rbcM) was randomly mutated and introduced under positive selection into Escherichia coli cells metabolically engineered to be dependent on Rubisco to detoxify its substrate ribulose 1,5-bisphosphate. Thirteen colonies displaying improved fitness were isolated, and all were found to harbor mutations in rbcM at one of two codons, histidine-44 or aspartate-117, that are structurally adjacent amino acids located about 10 A from the active site. Biochemical characterization of the mutant enzymes showed the mutations reduced their CO2/O2 specificity by 40% and decreased their carboxylation turnover rate by 20-40%. Structural analyses showed histidine-44 and aspartate-117 form a hydrogen bond in R. rubrum Rubisco and that the residues are conserved among other Form II Rubiscos. This study demonstrated the utility of directed evolution in E. coli for identifying catalytically relevant residues (in particular nonobvious residues disconnected from active site residues) and their potential molecular interactions that influence Rubisco's catalytic chemistry.
Publisher: Portland Press Ltd.
Date: 12-08-2008
DOI: 10.1042/BJ20080668
Abstract: The photosynthetic CO2-fixing enzyme Rubisco [ribulose-P2 (D-ribulose-1,5-bisphosphate) carboxylase/oxygenase] has long been a target for engineering kinetic improvements. Towards this goal we used an RDE (Rubisco-dependent Escherichia coli) selection system to evolve Synechococcus PCC6301 Form I Rubisco under different selection pressures. In the fastest growing colonies, the Rubisco L (large) subunit substitutions I174V, Q212L, M262T, F345L or F345I were repeatedly selected and shown to increase functional Rubisco expression 4- to 7-fold in the RDE and 5- to 17-fold when expressed in XL1-Blue E. coli. Introducing the F345I L-subunit substitution into Synechococcus PCC7002 Rubisco improved its functional expression 11-fold in XL1-Blue cells but could not elicit functional Arabidopsis Rubisco expression in the bacterium. The L subunit substitutions L161M and M169L were complementary in improving Rubisco yield 11-fold, whereas in idually they improved yield ∼5-fold. In XL1-Blue cells, additional GroE chaperonin enhanced expression of the I174V, Q212L and M262T mutant Rubiscos but engendered little change in the yield of the more assembly-competent F345I or F345L mutants. In contrast, the Rubisco chaperone RbcX stimulated functional assembly of wild-type and mutant Rubiscos. The kinetic properties of the mutated Rubiscos varied with noticeable reductions in carboxylation and oxygenation efficiency accompanying the Q212L mutation and a 2-fold increase in Kribulose-P2 (KM for the substrate ribulose-P2) for the F345L mutant, which was contrary to the ∼30% reductions in Kribulose-P2 for the other mutants. These results confirm the RDE systems versatility for identifying mutations that improve functional Rubisco expression in E. coli and provide an impetus for developing the system to screen for kinetic improvements.
Publisher: Springer Science and Business Media LLC
Date: 15-07-2008
Publisher: Informa UK Limited
Date: 23-07-2022
DOI: 10.1080/13506129.2022.2091985
Abstract: The study objective was to assess the effect of vutrisiran, an RNA interference therapeutic that reduces transthyretin (TTR) production, in patients with hereditary transthyretin (ATTRv) amyloidosis with polyneuropathy. HELIOS-A was a phase 3, global, open-label study comparing the efficacy and safety of vutrisiran with an external placebo group (APOLLO study). Patients were randomized 3:1 to subcutaneous vutrisiran 25 mg every 3 months (Q3M) or intravenous patisiran 0.3 mg/kg every 3 weeks (Q3W) for 18 months. HELIOS-A enrolled 164 patients (vutrisiran, Vutrisiran significantly improved multiple disease-relevant outcomes for ATTRv amyloidosis versus external placebo, with an acceptable safety profile. NCT03759379.
No related grants have been discovered for Bram De Wel.