“…We first identified several protein domains that have been reported to form oligomers of defined sizes in vitro ( Figures S1A and S1B ; Bolten et al, 2016 ; Boudko et al, 2009 ; Büttner et al, 2012 ; Chik et al, 2019 ; Drulyte et al, 2019 ; Huang et al, 2014 ; Lee et al, 1968 ; Li et al, 2019 ; Luo et al, 2001 ; Parsons et al, 2002 ; Santiago-Frangos et al, 2019 ; Sun et al, 2014 ; Thomson et al, 2014 ; Veesler et al, 2010 ). Each oligomerization domain was tagged with a fluorescent protein and expressed in C. elegans , and the size of oligomeric protein complexes from transgenic zygotes was examined using single-cell, single-molecule pull-down (sc-SiMPull) followed by photobleaching step counting ( Dickinson et al, 2017 ; Figure S1B ).…”
“…We first identified several protein domains that have been reported to form oligomers of defined sizes in vitro ( Figures S1A and S1B ; Bolten et al, 2016 ; Boudko et al, 2009 ; Büttner et al, 2012 ; Chik et al, 2019 ; Drulyte et al, 2019 ; Huang et al, 2014 ; Lee et al, 1968 ; Li et al, 2019 ; Luo et al, 2001 ; Parsons et al, 2002 ; Santiago-Frangos et al, 2019 ; Sun et al, 2014 ; Thomson et al, 2014 ; Veesler et al, 2010 ). Each oligomerization domain was tagged with a fluorescent protein and expressed in C. elegans , and the size of oligomeric protein complexes from transgenic zygotes was examined using single-cell, single-molecule pull-down (sc-SiMPull) followed by photobleaching step counting ( Dickinson et al, 2017 ; Figure S1B ).…”
“…It is one of three enzymes in the AraBAD operon that allow bacteria to utilize arabinose as an energy source by converting it into an intermediate in the pentose phosphate pathway ( d -Xu5P) (). Ru5P 4-epimerase is a homotetramer composed of four identical 25.5 kDa subunits and requires a divalent cation for activity ( − ). 1 Mechanism of the reaction catalyzed by l -ribulose- 5-phosphate 4-epimerase.…”
The structure of L-ribulose-5-phosphate 4-epimerase from E. coli has been solved to 2.4 A resolution using X-ray diffraction data. The structure is homo-tetrameric and displays C(4) symmetry. Each subunit has a single domain comprised of a central beta-sheet flanked on either side by layers of alpha-helices. The active site is identified by the position of the catalytic zinc residue and is located at the interface between two adjacent subunits. A remarkable feature of the structure is that it shows a very close resemblance to that of L-fuculose-1-phosphate aldolase. This is consistent with the notion that both enzymes belong to a superfamily of epimerases/aldolases that catalyze carbon-carbon bond cleavage reactions via a metal-stabilized enolate intermediate. Detailed inspection of the epimerase structure, however, indicates that despite the close overall structural similarity to class II aldolases, the enzyme has evolved distinct active site features that promote its particular chemistry.
“…The epimerase serves to link the arabinose metabolic pathway to the pentose phosphate pathway and allows the bacteria to use arabinose as an energy source (1). The enzyme has a molecular mass of 102 kDa (2,3), and is composed of four identical 25.5 kDa subunits (4,5). The epimerase is also known to require one divalent metal cation per subunit for activity (6).…”
Studies indicating that the E. coli L-ribulose-5-phosphate 4-epimerase employs an "aldolase-like" mechanism are reported. This NAD+-independent enzyme epimerizes a stereocenter that does not bear an acidic proton and therefore it cannot utilize a simple deprotonation-reprotonation mechanism. Sequence similarities between the epimerase and the class II l-fuculose-1-phosphate aldolase suggest that the two may be evolutionarily related and that the epimerization may occur via carbon-carbon bond cleavage and re-formation. Conserved residues thought to provide the metal ion ligands of the epimerase have been modified using site-directed mutagenesis. The resulting mutants show low kcat values in addition to a reduced affinity for Zn2+. These observations serve to establish that there is a structural link between between the active site geometry of the epimerase and the aldolase. In addition, the H97N mutant was found to catalyze the condensation of dihydroxyacetone and glycolaldehyde phosphate to produce a mixture of L-ribulose-5-phosphate and D-xylulose-5-phosphate. This observation of aldolase activity establishes that the epimerase active site is capable of promoting carbon-carbon bond cleavage. Furthermore, glycolaldehyde phosphate was shown to be a competitive inhibitor of the mutant enzyme (KI = 0.37 mM) but not of the wild-type enzyme. The mutation apparently causes the epimerase to become "leaky" and enables it to bind/generate the normal reaction intermediates from the unbound aldol cleavage products.
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