Nitrate reductase substrate

Sulfate reducers can use a wide range of terminal electron acceptors, and sulfate can be replaced by nitrate as a respiratory substrate. Molybdenum-containing enzymes have been discovered in SRB (also see later discussion) and, in particular, D. desulfuricans, grown in the presence of nitrate, generates a complex enzymatic system containing the following molybdenum enzymes (a) aldehyde oxidoreduc-tase (AOR), which reduces adehydes to carboxylic acids (b) formate dehydrogenase (FDH), which oxidizes formate to CO2 and (c) nitrate reductase (the first isolated from a SRB), which completes the enzy-... 

So little is known about molybdenum enzymes other than milk xanthine oxidase that there is little to be said by way of general conclusions. In all cases where there is direct evidence (except possibly for xanthine dehydrogenase from Micrococcus lactilyticus) it seems that molybdenum in the enzymes does have a redox function in catalysis. For the xanthine oxidases and dehydrogenases and for aldehyde oxidase, the metal is concerned in interaction of the enzymes with reducing substrates. However, for nitrate reductase it is apparently in interaction with the oxidizing substrate that the metal is involved. In nitrogenase the role of molybdenum is still quite uncertain. 

Fig. 1. The nitrate assimilation pathway in higher plants. The pathway of nitrate assimilation in the tobacco leaf is illustrated. In some other species an additional cytosolic GS is found in the leaf. The pathway in plant roots is more poorly documented and more variable GS in roots is mostly cytosolic, and some enzymes such as GOGAT are found as isoforms utilising alternate reducing substrates. T, expected nitrate carrier NR, nitrate reductase NiR, nitrite reductase GS, glutamine synthetase GOGAT, glutamate synthase Fd, ferredoxin Gin, glutamine Glu, glutamate.

Although molybdenum and tungsten enzymes carry the name of a single substrate, they are often not as selective as this nomenclature suggests. Many of the enzymes process more than one substrate, both in vivo and in vitro. Several enzymes can function as both oxidases and reductases, for example, xanthine oxidases not only oxidize purines but can deoxygenate amine N-oxides [82]. There are also sets of enzymes that catalyze the same reaction but in opposite directions. These enzymes include aldehyde and formate oxidases/carboxylic acid reductase [31,75] and nitrate reductase/nitrite oxidase [83-87]. These complementary enzymes have considerable sequence homology, and the direction of the preferred catalytic reaction depends on the electrochemical reduction potentials of the redox partners that have evolved to couple the reactions to cellular redox systems and metabolic requirements. 

Given the notion of microscopic reversibility, and the similarity in the active sites of sulfite oxidase and nitrate reductase (assimilatory), determining the mechanism of action of sulfite oxidase impacts upon our understanding of reductases in the (MPT)Mo(0)2 family. A key issue in the mechanism of sulfite oxidase is whether substrate binds to the metal center during the catalytic cycle. Substrate (or product) binding to the molybdenum center, as proposed for the catalytic... 

More studies of the sulfite oxidase/nitrate reductase catalytic cycles, especially those using carefully isotopically labeled enzyme and substrate, are required before a clear mechanistic picture for (MPT)Mo(0)2(S-cys) family of enzymes will be available. 

Similar mechanisms operate in the action of nitrate reductase and nitrite reductase. Both of these substances are produced from ammonia by oxidation. Plants and soil bacteria can reduce these compounds to provide ammonia for metabolism. The common agricultural fertilizer ammonium nitrate, NH4NO3, provides reduced nitrogen for plant growth directly, and by providing a substrate for nitrate reduction. NADH or NADPH is the electron donor for nitrate reductase, depending on the organism. 

Nitrate Reductase Activity. There are similarities between induced nitrate reductase activity and induced iron stress response. In both, biochemical reactions are induced, and a substrate is reduced N03 to N02 by nitrate reductase and Fe3+ to Fe2+ by a reductant activated in response to iron stress. Chemical reactions induced by iron stress increased the use of iron, and simultaneously increased nitrate reductase activity in roots (Figure 5) and in tops of iron-efficient tomato. This induced nitrate reductase activity declined when iron was made available to the plants. 

A cycle for substrate reduction can be devised by beginning at the reduced Mo(IV) state and reversing the direction of OAT and CEPT steps, as illustrated for the nitrate reductase reaction in Fig. 9(b). Electrons are provided to reduce Mo(VI) to Mo(IV) via cytochrome b and FAD. 

The above schemes work reasonably well for certain enzyme reactions, especially for substrates where oxygen addition/loss occurs at a main group element (e.g., N, S, Se, Cl, see Table I). In addition to SO and nitrate reductase, key examples are DMSOR, trimethylamine oxide reductase, chlorate reductase, and selenate reductase. In the case of enzymes catalyzing C-based redox reactions of organic molecules, notably XDH and aldehyde oxidase, a direct OAT step is unlikely and is replaced by mechanistic steps typical of hydro-xylation (2). The essential features of the mechanism are shown in Fig. 10 for xanthine dehydrogenase/oxidase. 

Enzymes in this family include DMSO reductase, biotin 5-oxide reductase, dissimilatory nitrate reductase, trimethylamine A-oxide reductase, and formate dehydrogenase they are found exclusively in bacteria and fungi and act as terminal respiratory reductases during anaerobic growth in the presence of their respective substrates. " DMSO reductases catalyze the reaction shown in equation (5) the water-soluble enzymes from the purple phototrophic bacteria R. capsula-tus and R. sphaeroides are among the simplest Mo-MPT enzymes, being relatively small (ca. 85 kDa), single subunit... 

Lomas, M. W. (2004b). Nitrate reductase and urease enzyme activity in the marine diatom Thalassiosira weissjlogii (BaciUariophyceae) Interactions among nitrogen substrates. Mar. Biol. 144, 37 4. 

These results clearly indicate that by disrupting the nitrate reductase system, an Ecoli strain suitable for practical hydrogen production can be developed which will not be affected by the presence of nitrate in the utilizable substrate. The availability of a mutant strain enabled us to demonstrate the application of a mutant strain deficient in the narG locus. However, the existence of a constitutively synthesized nitrate reductase was reported, a nitrate reductase Z (10). Indeed, by the cultivation of the strain RK5265 in the presence of 100 mM nitrate, no FHL activity was observed (results not shown). Therefore, the ideal E.coli strain will be constructed by introducing mutation in the structural gene for FDH-N. 

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