期刊
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
卷 71, 期 5, 页码 2473-2478出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/AEM.71.5.2473-2478.2005
关键词
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Oxidative cleavage of poly(cis-1,4-isoprene) by rubber oxygenase RoxA purified from Xanthomonas sp. was investigated in the presence of different combinations of O-16(2), O-18(2), (H2O)-O-16, and (H2O)-O-18. 12-Oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD; m/z 236) was the main cleavage product in the absence of O-18-compounds. Incorporation of one 18 0 atom in ODTD was found if the cleavage reaction was performed in the presence of O-18(2) and (H2O)-O-16. Incubation of poly (cis-1,4-isoprene) (with RoxA) or of isolated unlabeled ODTD (without RoxA) with (H2O)-O-18 in the presence of 16 02 indicated that the carbonyl oxygen atoms of ODTD significantly exchanged with oxygen atoms derived from water. The isotope exchange was avoided by simultaneous enzymatic reduction of both carbonyl functions of ODTD to the corresponding dialcohol (12-hydroxy-4,8-dimethyltrideca-4,8-diene-1-ol (HDTD; m/z 240) during RoxA-mediated in vitro cleavage of poly (cis-1,4-isoprene). In the presence of O-18(2), (H2O)-O-16, and alcohol dehydrogenase/NADH, incorporation of two atoms of O-18 into the reduced metabolite HDTD was found (m/z 244), revealing that RoxA cleaves rubber by a dioxygenase mechanism. Based on the labeling results and the presence of two hemes in RoxA, a model of the enzymatic cleavage mechanism of poly(cis-1,4-isoprene) is proposed.
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