Structure of the glycosyltransferase EryCIII in complex with its activating P450 homologue EryCII.
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EryCII forms a heterotetramer with EryCIII, stabilizes it, and acts as an allosteric activator of the glycosyltransferase.
"Our biophysical and enzymatic data support a model in which EryCII stabilizes EryCIII and also functions as an allosteric activator of the GT."
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The EryCIII-EryCII complex is a heterotetramer.
"The structure reveals a heterotetramer with a distinctive, elongated quaternary organization."
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EryCII lacks the conserved heme-ligating cysteine of P450s and has no heme; it is not an active P450 enzyme (structure-based alignment, apo structure).
"As expected for EryCII—and indeed the macrolide auxiliary proteins generally—the conserved cysteine is absent (Supplementary Fig. S5); thus, they are not active P450 enzymes."
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EryCII has lost the heme group from the central core of the P450-like fold.
"Having lost the heme group in the central core, the protein may be more conformationally dynamic"
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EryCIII is inactive on its own; EryCII is required to restore its glycosyltransferase activity.
"When separate EryCIII- and EryCII-containing extracts were combined, the GT activity of EryCIII was restored, confirming as expected that EryCII is required by EryCIII."
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The complex is an alpha2-beta2 heterotetramer (dimer of EryCIII-EryCII heterodimers); EryCII activates allosterically via its N-terminal A'' helix, which contacts EryCIII's acceptor domain without touching the active site directly.
"Binding of EryCII to the N-terminal acceptor domain of EryCIII (Fig. 6) may likewise trigger a closer association with the C-terminal sugar-nucleotide binding domain and the correct juxtaposition of the acceptor and donor sites of EryCIII."