The lipopolysaccharide of Yersinia pseudotuberculosis V includes a 3,6-dideoxyhexose, ascarylose, as the nonreducing end of the O-antigen tetrasaccharide. The C-3 deoxygenation of CDP-6-deoxy-L-threo-D-glycero-4-hexulose is a critical reaction in the biosynthesis of ascarylose. The first half of the reaction is a dehydration catalyzed by CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), which is PMP-dependent and contains a redox-active [2Fe-2S] center. The second half is a reduction that requires an additional enzyme, CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3, formerly known as CDP-6-deoxy-delta 3,4-glucoseen reductase), which has a FAD and a [2Fe-2S] center in the active site. Using NADH as the reductant in the coupled E1-E3 reaction, we have monitored the kinetics of a radical intermediate using both stopped-flow spectrophotometry and rapid freeze-quench EPR under aerobic and hypoxic conditions. In the EPR studies, a sharp signal at g = 2.003 was found to appear at a rate which is kinetically competent, reaching its maximum intensity at approximately 150 ms. Stopped-flow UV-vis analysis of the reaction elucidated a minimum of six optically distinguishable states in the mechanism of electron transfer from NADH to substrate. Interestingly, one of the detected intermediates has a time course nearly identical to that of the radical detected by rapid freeze-quench EPR. The difference UV-vis spectrum of this intermediate displays a maximum at 456 nm with a shoulder at 425 nm. Overall, these results are consistent with an electron transfer pathway that includes a radical intermediate with the unpaired spin localized on the substrate-cofactor complex. Evidence in support of this mechanism is presented in this report. These studies add the PMP-glucoseen radical to the growing list of mechanistically important bioorganic radical intermediates that have recently been discovered.

The conversion of CDP-4-keto-6-deoxy-D-glucose to CDP-4-keto-3,6-dideoxy-D-glucose is a key step in biosynthesis of ascarylose, the terminal dideoxyhexose of the O-antigen tetrasaccharide of the lipopolysaccharide from Yersinia pseudotuberculosis V. This transformation is catalyzed by two enzymes: CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase (E1), which contains a pyridoxamine and a [2Fe-2S] center, and an NADH-dependent CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydrase reductase (E3), which contains both an FAD and a [2Fe-2S] center. E1 reacts to form a Schiff base with CDP-4-keto-6-deoxy-D-glucose and catalyzes the elimination of the hydroxyl at position 3 of the glucose moiety, resulting in the formation of a covalently bound CDP-6-deoxy-delta(3,4)-glucoseen intermediate. E3 transfers electrons from NADH to E1, which uses these to reduce the delta(3,4)-glucoseen bond to produce CDP-4-keto-3,6-dideoxy-D-glucose. In this work, we have investigated the reductive half-reaction of E3 using both single wavelength and diode array stopped flow absorbance spectroscopy. We find that NADH binds to both oxidized (Kd = 52.5 +/- 2 microM) and two-electron-reduced (Kd = 12.1 +/- 1 microM) forms of E3. Hydride transfer from NADH to the FAD moiety occurs at 107.5 +/- 3 s-1 and exhibits a 10-fold deuterium isotope effect when (4R)-[2H]NADH is substituted for NADH. Following the hydride transfer reaction, NAD+ is released at 42.5 +/- 1 s-1 and electron transfer from the reduced FAD to the [2Fe-2S] center occurs rapidly. The extent of the intramolecular electron transfer reaction is pH-dependent with a pKa of 7.3 +/- 0.1, which may represent the ionization state of the N-1 position of the FAD hydroquinone of E3. Finally, E3 is converted to the three-electron-reduced state in a slow disproportionation reaction that consumes NADH: The [2Fe-2S] center of E3 was selectively disassembled by titration with mersalyl to give E3(apoFeS). The properties of this form of the enzyme are compared to those of the holoenzyme. Similarities and differences of the reductive half-reactions of E3 and related iron-sulfur flavoenzymes are discussed.

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In an effort to characterize the diversity of mechanisms involved in cellular self-protection against the antitumor antibiotic mitomycin C (MC), DNA fragments from the producing organism (Streptomyces lavendulae) were introduced into Streptomyces lividans and transformants were selected for resistance to the drug. Subcloning of a 4.0-kb BclI fragment revealed the presence of an MC resistance determinant, mrd. Nucleotide sequence analysis identified an open reading frame consisting of 130 amino acids with a predicted molecular weight of 14,364. Transcriptional analysis revealed that mrd is expressed constitutively, with increased transcription in the presence of MC. Expression of mrd in Escherichia coli resulted in the synthesis of a soluble protein with an M r of 14,400 that conferred high-level cellular resistance to MC and a series of structurally related natural products. Purified MRD was shown to function as a drug-binding protein that provides protection against cross-linking of DNA by preventing reductive activation of MC.Streptomyces species are gram-positive soil bacteria known for their ability to produce a wide range of biologically active metabolites. In addition, many resistance genes have been cloned from these bacteria. Mechanisms of cellular self-protection include drug inactivation, target site modification, reduction of intracellular concentration via efflux, and drug binding (8). The presence of multiple modes of self-protection toward a single antibiotic is well documented (9), often with one or more resistance determinants located adjacent to the corresponding biosynthetic genes.Mitomycin C (MC) was identified in 1956 as an antibiotic produced by Streptomyces lavendulae (18) and subsequently established as an important antitumor agent (19,21). MC functions as a prodrug and requires enzymatic or chemical reduction to become a highly reactive alkylating agent (19,40). The intracellular activation of MC is specified by endogenous flavoreductases (34) and proceeds by single electron reduction to the MC semiquinone radical. The relatively long-lived semiquinone species either rearranges to an alkylating intermediate (by further reduction) or transfers an electron to molecular oxygen to generate superoxide (32). Therefore, the ability of MC to inhibit bacterial and mammalian cell growth involves the combined action of DNA alkylation and the formation of reactive oxygen species. Other naturally occurring compounds within this class include bleomycin (37), enediynes (10), and the more recently discovered dihydrobenzoxazines (25,42).Recently, a locus, mcr, that confers high-level MC resistance in S. lavendulae has been reported (2, 3). The resistance gene, mcrA, encodes a flavoenzyme (MCRA) that reoxidizes reductively activated MC (23). In another example involving a DNA damaging agent, bleomycin self-resistance has been determined by drug modification (Bat) and binding (BLMA) proteins in Streptomyces verticillus (38). Beyond these examples, little is known about bacterial resistance to the growing cl...

Many biological transformations are effected via electron transfer or homolytic bond cleavage, and thus proceed through radical mechanisms. 1 Early studies of the biosynthesis of immunodominant 3,6-dideoxyhexoses found in the lipopolysaccharide of several pathogenic bacterial strains, as exemplified by the biosynthesis of CDP-L-ascarylose (1) in Yersinia pseudotuberculosis V, have demonstrated that the C-3 deoxygenation step proceeds through a radical mechanism and requires a unique pair of enzymes, E 1 2 and E 3 . 2,3 E 1 contains a [2Fe-2S] center and requires pyridoxamine 5′-phosphate (PMP). 4 E 3 contains a flavin and a [2Fe-2S] center and uses NADH as a reductant. 5 In the present work, isotopic labeling of PMP has been combined with EPR techniques to provide unambiguous evidence of a radical being directly associated with the PMP coenzyme in E 1 catalysis.The catalytic cycle for deoxygenation begins with formation of the Schiff base (3) between substrate 2 and PMP in the active site of E 1 (Scheme 1). Subsequent proton abstraction triggers the elimination of 3-OH to give 4. 4 Transfer of reducing equivalents from NADH via E 3 to reduce the nascent ∆ 3,4 -glucoseen intermediate 4 completes the reaction. 5 Previous studies have revealed the presence of a flavin semiquinone radical in E 3 6 and another organic radical in E 1 7 during transient phases of the reaction. Recent studies led to a model in which the odd electron in the half-reduced intermediate resides primarily on the PMP portion of the PMP-substrate adduct, perhaps as a phenoxyl radical (5a). 7To characterize this organic radical, the isotopically labeled forms of PMP, [4′,5′-2 H 4 ]PMP (6), and [2′-2 H 3 ]PMP (7) have been prepared. 8 Each labeled PMP was used to reconstitute E 1 -(apoPMP), 9 and each reconstituted E 1 was used in the coupled E 1 -E 3 reaction to prepare samples for CW EPR and pulsed electron nuclear double resonance (pulsed ENDOR) measurements. 10 Figure 1 (inset) shows the CW EPR spectrum of E 1 reconstituted with 6 (heavy line). The signal from the sample with deuterated PMP narrows by approximately 3 G compared with the reference spectrum using E 1 reconstituted with unlabeled PMP (light line). The spin concentration of this organic radical was estimated to be 7.5 µM, which was significantly higher than the maximum possible flavin semiquinone concentration of 0.4 µM calculated on the basis of E 3 concentration in the E 1 -E 3 reaction mixture. The observed sharpening effect on the EPR signal is Lei, Y.; Vatanen, K.; Liu, H.-w. Biochemistry 1995, 34, 4159-4168. (d) Burns, K. D.; Pieper, P. A.; Liu, H.-w.; Stankovich, M. T. Gassner, G. T.; Bandarian, V.; Ruzicka, F. J.; Ballou, D. P.; Reed, G. H.; Liu, H.-w. Biochemistry 1996, 35, 15846-15856. (8) Compound 6 was prepared based primarily on an established scheme (Pieper, P. A.; Yang, D.-y.; Zhou, Z.-q.; Liu, H.-w. J. Am. Chem. Soc. 1997, 119, 1809-1817. Preparation of 7 also followed a previously developed sequence (Yang, D.-y.; Shih, Y.; Liu, H.-w. J. Org. Chem. 1991, 56, 294...

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The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduced and activated intracellularly, producing cytotoxic semiquinone anion radical and hydroquinone reduction intermediates. In vitro, MCRA protects DNA from crosslinking by the hydroquinone reduction intermediate of these mitomycins by oxidizing the hydroquinone back to the parent molecule; thus, MCRA acts as a hydroquinone oxidase. These findings suggest potential therapeutic applications for MCRA in the treatment of cancer with the mitomycins and imply that intrinsic or selected mitomycin C resistance in mammalian cells may not be due solely to decreased bioactivation, as has been hypothesized previously, but instead could involve an MCRA-like mechanism.

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