Structure of the processive rubber oxygenase RoxA from Xanthomonas sp

Abstract

Rubber oxygenase A (RoxA) is one of only two known enzymes able to catalyze the oxidative cleavage of latex for biodegradation. RoxA acts as a processive dioxygenase to yield the predominant product 12-oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD), a tri-isoprene unit. Here we present a structural analysis of RoxA from Xanthomonas sp. strain 35Y at a resolution of 1.8 Å. The enzyme is a 75-kDa diheme c -type cytochrome with an unusually low degree of secondary structure. Analysis of the heme group arrangement and peptide chain topology of RoxA confirmed a distant kinship with diheme peroxidases of the CcpA family, but the proteins are functionally distinct, and the extracellular RoxA has evolved to have twice the molecular mass by successively accumulating extensions of peripheral loops. RoxA incorporates both oxygen atoms of its cosubstrate dioxygen into the rubber cleavage product ODTD, and we show that RoxA is isolated with O 2 stably bound to the active site heme iron. Activation and cleavage of O 2 require binding of polyisoprene, and thus the substrate needs to use hydrophobic access channels to reach the deeply buried active site of RoxA. The location and nature of these channels support a processive mechanism of latex cleavage.

RoxB Is a Novel Type of Rubber Oxygenase That Combines Properties of Rubber Oxygenase RoxA and Latex Clearing Protein (Lcp)

“…Carbon monoxide is able to bind to RoxA Xsp in the form as isolated, as revealed by the CO-dependent appearance of an absorption peak at 415 nm in the difference spectrum (21). This binding can be explained by the CO affinity of the reduced, oxygen-bearing heme center (10). Carbon monoxide strongly inhibits the polyisoprene cleavage reaction of RoxA Xsp (21).…”

“…Production and excretion of RoxB Xsp , containing two costly cofactors, would not seem advantageous for the bacteria if they are not able to use the reaction products for metabolism. One reason for the presence of a roxB gene might be the fact that RoxA Xsp is an exo-type cleavage enzyme that needs free polyisoprene ends to cleave the polymer in a processive manner to give the only observed end product, ODTD (10). The presence of RoxB Xsp , which cleaves rubber in an endo-type fashion to a mixture of oligo-isoprenoids, would enlarge the number of poly/oligo-isoprenoid chains with free, accessible ends.…”

“…RoxA Xsp was identified as a c-type diheme dioxygenase (7)(8)(9). Recently, the three-dimensional RoxA Xsp structure and essential amino acids of the active site were determined (10,11). RoxA homologs were identified in other Gram-negative bacteria such as Haliangium ochraceum and some myxobacteria (12) but not in any Grampositive rubber degrader.…”

Latex Clearing Protein (Lcp) of Streptomyces sp. Strain K30 Is a b -Type Cytochrome and Differs from Rubber Oxygenase A (RoxA) in Its Biophysical Properties

“…Previous structural and biophysical analysis had revealed that the heme group of the RoxA active site has a dioxygen molecule bound to the heme iron atom of the active heme site (11,13). Because of the strong electronegativity of oxygen, one electron of the Fe 2ϩ ion is relocated to the dioxygen molecule, resulting in an Fe 3ϩ -O 2 Ϫ state.…”

“…strain 35Y (9). Biochemical and biophysical investigation revealed that RoxA is an extracellular dioxygenase with two covalently attached heme groups (10,11) and is structurally but not functionally related to cytochrome c peroxidases (12,13). RoxA cleaves poly(cis-1,4-isoprene) to 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD; C 15 -tri-isoprenoid) as a main end product.…”