Structural basis for the different cleavage mechanisms of poly(1,4-cis-isoprene) by rubber oxygenases RoxA and RoxB

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Steroidobacter cummioxidans 35Y (previously Xanthomonas sp. 35Y) forms two related, synergistically acting, extracellular haem-containing rubber (di)oxygenases, RoxA and RoxB, during growth on rubber. RoxA cleaves rubber processively to the C15 oligoisoprenoid 12-oxo-4,8-dimethyl-tri-deca-4,8-diene-1-al (ODTD) in an exo-type fashion, and RoxB cleaves rubber to C20 and higher oligoisoprenoids randomly in an endo-type manner. The 3D structure of RoxA was used to model the structure of RoxB. While the cores of RoxA and RoxB are almost identical they differ in three major loops that form a “dome” above the active haem site. The dome must undergo a conformational change to allow access of the substrate (polyisoprene chain) to the active site haem. We postulate that the differences in the dome-loops between RoxA and RoxB are responsible for the different cleavage modes and cleavage products in RoxA and RoxB. We will address this hypothesis by (i) determination of the (true) RoxB 3D structure, (ii) by biochemical and biophysical analysis of chimeric RoxA-RoxB variants (both constructed chimers and naturally occurring RoxA/RoxB hybrids (RoxCs) and sited-directed mutated rubber oxygenase variants) and (iii) by analysis of the substrate-protein interaction with Bio Layer Interferometrie.