Structural and Biochemical Identification of a Novel Bacterial Oxidoreductase

Abstract

By using a bioinformatics screen of the Escherichia coli genome for potential molybdenum-containing enzymes, we have identified a novel oxidoreductase conserved in the majority of Gram-negative bacteria. The identified operon encodes for a proposed heterodimer, YedYZ in Escherichia coli, consisting of a soluble catalytic subunit termed YedY, which is likely anchored to the membrane by a heme-containing trans-membrane subunit termed YedZ. YedY is uniquely characterized by the presence of one molybdenum molybdopterin not conjugated by an additional nucleotide, and it represents the only molybdoenzyme isolated from E. coli characterized by the presence of this cofactor form. We have further characterized the catalytic subunit YedY in both the molybdenum-and tungsten-substituted forms by using crystallographic analysis. YedY is very distinct in overall architecture from all known bacterial reductases but does show some similarity with the catalytic domain of the eukaryotic chicken liver sulfite oxidase. However, the strictly conserved residues involved in the metal coordination sphere and in the substrate binding pocket of YedY are strikingly different from that of chicken liver sulfite oxidase, suggesting a catalytic activity more in keeping with a reductase than that of a sulfite oxidase. Preliminary kinetic analysis of YedY with a variety of substrates supports our proposal that YedY and its many orthologues may represent a new type of membrane-associated bacterial reductase.Molybdenum-coordinating enzymes fall within the broad class of enzymes associated with redox metabolic functions in prokaryotic and eukaryotic cells. The structurally characterized enzymes can be roughly grouped into three separate families (the bacterial/eukaryotic xanthine oxidase family, the eukaryotic sulfite oxidase family, and the bacterial Me 2 SO reductase family), each distinctive with respect to active site structure and the type of reaction they catalyze (1). The family of xanthine oxidases contains 1 eq of a pterin cofactor coordinated to the molybdenum metal with the typical pentavalent, approximately octahedral coordination sphere in the oxidized state completed not by any side chains from the enzyme but rather by a double-bonded sulfur atom, a double-bonded oxygen atom, and an oxygen atom with a single bond (2). Sulfite oxidases have 1 eq of a pterin cofactor with the molybdenum coordinated by a cysteine ligand from the enzyme and two oxo groups (3, 4). Kappler et al. (5) described the spectroscopic and enzymologic characterization of a member of the sulfite oxidase family from Thiobacillus novellus, and they showed that the enzyme contains a molybdopterin-type cofactor, but no structural data are available for bacterial sulfite oxidase family members to verify the nature of the cofactor or overall architecture of this enzyme. The Me 2 SO reductase family is diverse in both structure and function, but all members have 2 eq of the pterin cofactor, and the molybdenum coordination sphere is usually completed by a single oxo group ...

Molybdenum cofactor-dependent resistance to N-hydroxylated base analogs in Escherichia coli is independent of MobA function

“…E. coli contains only a few, largely putative, members of the last three families: YdhV, YedY, and the three putative xanthine dehydrogenases XdhA, XdhD, and YagR (Table 1). Among these, the crystal structure of YedY has indeed been shown the presence of MPT as cofactor, but the activity and cellular function of the protein are unclear at this time [30,37]. The three putative xanthine oxidases may play a role in purine catabolism, although their main function remains to be determined [23].…”

“…The latter two have been identified as alternative DMSO reductases [29]. The database searches also identified one putative member of the sulfite oxidase family, YedY [30], one putative member of the aldehyde ferredoxin oxidoreductase family, YdhV, and three putative xanthine oxidases, XdhA, XdhD, and YagR [23].…”

Electrochemical evidence that pyranopterin redox chemistry controls the catalysis of YedY, a mononuclear Mo enzyme

“…YedY has been structurally characterized via both X-ray crystallography and X-ray absorption spectroscopy (XAS) (3,8,9). In most mononuclear Mo enzymes, heme groups and iron sulfur clusters are found within the same protein as the Mo center, but the only metal site in YedY is Mo, making this enzyme a helpfully simple system for studying redox chemistry ( Fig.…”

“…The general ability of mononuclear Mo enzymes to catalyze two-electron oxygen atom transfer reactions has been attributed to the Mo(IV)/ Mo(V)/Mo(VI) oxidation state cycling of the active site, and this mechanism is a common part of undergraduate syllabuses (1,2). Escherichia coli YedY is a mononuclear Mo enzyme (3), and based on sequence homology, the majority of sequenced Gramnegative bacterial genomes encode a YedY-like protein (3)(4)(5). Uniquely for a mononuclear Mo enzyme, it has not been possible to form the YedY Mo(VI) state in experiments using ferricyanide as an oxidizing agent, and an unusually positive reduction potential for the Mo(V/IV) transition [+132 mV vs. standard hydrogen electrode (SHE) at pH 7] was determined from EPR experiments (6).…”

“…Although the physiological substrate of YedY is unknown, a possible role in the reduction of reactive nitrogen species is suggested by experiments on the pathogen Campylobacter jejuni, where deletion of the Cj0379 YedY homolog generated a mutant that is deficient in chicken colonization and has a nitrosative stress phenotype (4). YedY catalysis can be assayed by measuring the two-electron reduction of either dimethyl sulfoxide (DMSO) or trimethylamine N-oxide (TMAO) (3), but the inaccessibility of the YedY Mo(VI) means the enzyme mechanism does not proceed via the common two-electron Mo-redox cycle. In small-molecule analogs of mononuclear Mo enzymes, the pterin ligands are described as "noninnocent," meaning that the redox processes could be ligand or metal based (7).…”