Enzymatic properties and effect of ionic strength on periplasmic nitrate reductase (NAP) from Desulfovibrio desulfuricans ATCC 27774.
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).
Bursakov
S A
SA
Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Monte de Caparica, Portugal.
Carneiro
C
C
Almendra
M J
MJ
Duarte
R O
RO
Caldeira
J
J
Moura
I
I
Moura
J J
JJ
D017738:Alkanesulfonic Acids; D000595:Amino Acid Sequence; D002021:Buffers; D004396:Coloring Agents; D003901:Desulfovibrio / Q000201:enzymology*; D004355:Drug Stability; D004789:Enzyme Activation / Q000187:drug effects; D004983:Ethanolamines; D005292:Ferricyanides / Q000494:pharmacology; D008969:Molecular Sequence Data; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism; D009994:Osmolar Concentration; D019897:Periplasm / Q000201:enzymology*; D011190:Potassium Cyanide / Q000494:pharmacology; D014755:Viologens / Q000378:metabolism
D017738:Alkanesulfonic Acids; D002021:Buffers; D004396:Coloring Agents; D004983:Ethanolamines; D005292:Ferricyanides; D009566:Nitrates; D014755:Viologens; C007931:hexacyanoferrate III; C103648:3-((1,1-dimethyl-2-hydroxyethyl)amino)-2-hydroxypropanesulfonic acid; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D011190:Potassium Cyanide