FIG. 6.

NAD⁺-dependent HGMB conversion by the dehydrogenase of Rhodococcus sp. strain AD45. HPLC elution profiles of assays without dehydrogenase (A) and with dehydrogenase (70 μg/ml) (B). The assay mixture consisted of 0.4 mM NAD⁺ and 0.4 mM HGMB in 50 mM glycine-NaOH (pH 10). Activity was monitored by following the generation of NADH at 340 nm, and samples for HPLC-MS analysis were removed at 60 min. Compounds 1 (HGMB) and 2 were identified as conjugates of GSH and isoprene monoxide. Compound 3 was identified as 2-glutathionyl-2-methyl-3-butenoic acid.