Enzymatic Properties and Effect of Ionic Strength on Periplasmic Nitrate Reductase (NAP) fromDesulfovibrio desulfuricansATCC 27774
Abstract
Some sulfate reducing bacteria can induce nitrate reductase when grown on nitrate containing media being involved in dissimilatory reduction of nitrate, an important step of the nitrogen cycle. Previously, it was reported the purification of the first soluble nitrate reductase from a sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774 (S.A. Bursakov, M.-Y. Liu, W.J. Payne, J. LeGall, I. Moura, and J.J.G. Moura (1995) Anaerobe 1, 55-60). The present work provides further information about this monomeric periplasmic nitrate reductase (Dd NAP). It has a molecular mass of 74 kDa, 18.6 U specific activity, KM (nitrate) = 32 microM and a pHopt in the range 8-9.5. Dd NAP has peculiar properties relatively to ionic strength and cation/anion activity responses. It is shown that monovalent cations (potassium and sodium) stimulate NAP activity and divalent (magnesium and calcium) inhibited it. Sulfate anion also acts as an activator in KPB buffer. NAP native form is protected by phosphate anion from cyanide inactivation. In the presence of phosphate, cyanide even stimulates NAP activity (up to 15 mM). This effect was used in the purification procedure to differentiate between nitrate and nitrite reductase activities, since the later is effectively blocked by cyanide. Ferricyanide has an inhibitory effect at concentrations higher than 1 mM. The N-terminal amino acid sequence has a cysteine motive C-X2-C-X3-C that is most probably involved in the coordination of the [4Fe-4S] center detected by EPR spectroscopy. The active site of the enzyme consists in a molybdopterin, which is capable for the activation of apo-nit-1 nitrate reductase of Neurospora crassa. The oxidized product of the pterin cofactor obtained by acidic hidrolysis of native NAP with sulfuric acid was identified by HPLC chromatography and characterized as a molybdopterin guanine dinucleotide (MGD).
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The detailed kinetic properties of the monomeric NapA from D. desulfuricans have revealed that ionic strength has a peculiar effect on the activity.
“The detailed kinetic properties of the monomeric NapA from D. desulfuricans have revealed that ionic strength has a peculiar effect on the activity.”
From Nitrate and periplasmic nitrate reductases (2014) · cites this paper as [210]
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- Authors
- Bursakov, S. A., Carneiro, C., Almendra, M. J., Duarte, R. O., Caldeira, J., Moura, I., & Moura, J. J. G.
- Affiliations
- A N Bach Institute of Biochemistry (RU)
- Universidade Nova de Lisboa (PT)
- Venue
- Biochemical and Biophysical Research Communications
- Publisher
- Elsevier BV
- Published
- 1997-10
- Volume
- 239
- Issue
- 3
- Pages
- 816-822
- ISSN
- 0006-291X (ISSN-L)
- OpenAlex
- W2027558062
- Cited by
- 42
- References
- 49
- Citations by year
- 2025: 1 · 2024: 1 · 2023: 4 · 2022: 2 · 2020: 2 · 2019: 1 · 2018: 1 · 2017: 1 · 2015: 1 · 2014: 3 · 2013: 1 · 2012: 1
- FWCI
- 2.81
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biochemical and Biophysical Research Communications (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Metalloenzymes and iron-sulfur proteins
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
- Physical Sciences › Environmental Science › Pollution › Wastewater Treatment and Nitrogen Removal
- Keywords
- periplasmic nitrate reductase, Desulfovibrio desulfuricans, sulfate-reducing bacteria, dissimilatory nitrate reduction, ionic strength effects, cation activation, sulfate activation, molybdopterin, [4Fe-4S] cluster, cysteine motifs, enzyme kinetics, enzyme purification
- MeSH terms
- Amino Acid Sequence, Buffers, Desulfovibrio, Drug Stability, Coloring Agents, Enzyme Activation, Ethanolamines, Ferricyanides, Molecular Sequence Data, Nitrate Reductases, Nitrates, Osmolar Concentration, Potassium Cyanide, Viologens, Alkanesulfonic Acids, Periplasm, Nitrate Reductase
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