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Properties of the selenium- and molybdenum-containing nicotinic acid hydroxylase from Clostridium barkeri.
Biochemistry. 1996 Jan 09; 35(1):212-23.B

Abstract

NADP(+)-coupled nicotinic acid hydroxylase (NAH) has been purified to near-homogeneity from Clostridium barkeri by an improved purification scheme that allowed the isolation of milligram amounts of enzyme of higher specific activity then previously reported. NAH is most stable at alkaline pH in the presence of glycerol. The protein which consists of four dissimilar subunits occurs in forms of different molecular masses. There are 5-7 Fe, 1 FAD, and 1 Mo per 160 kDa protein promoter. Mo in the enzyme is bound to a dinucleotide form of molybdopterin and is coordinated with selenium. Mo(V), flavin radical, and two Fe2S2 clusters could be observed with EPR spectroscopy. The Se cofactor which is essential for nicotinic acid hydroxylase activity could be released from NAH as a reactive low molecular weight compound by a number of denaturing procedures. Parallel losses of Se and catalytic activity were observed during purification and storage of the enzyme. Addition of sodium selenide or selenophosphate did not restore the catalytic activity of the enzyme. Instead, NAH is reversibly inactivated by these compounds and also by sulfide. Cyanide, a common inhibitor of Mo-containing hydroxylases, does not affect NAH catalytic activity. The "as isolated" enzyme exhibits a Mo(V) EPR signal (2.067 signal) that was detected at early stages of purification. NAH exhibits a high substrate specificity toward electron donor substrates. The ability of a nicotinate analog to reduce NAH (disappearance of 2.067 signal) correlates with the rate of oxidation of the analog in the standard assay mixture. The properties of NAH differentiate the enzyme from known Mo-containing hydroxylases.

Authors+Show Affiliations

Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

8555176

Citation

Gladyshev, V N., et al. "Properties of the Selenium- and Molybdenum-containing Nicotinic Acid Hydroxylase From Clostridium Barkeri." Biochemistry, vol. 35, no. 1, 1996, pp. 212-23.
Gladyshev VN, Khangulov SV, Stadtman TC. Properties of the selenium- and molybdenum-containing nicotinic acid hydroxylase from Clostridium barkeri. Biochemistry. 1996;35(1):212-23.
Gladyshev, V. N., Khangulov, S. V., & Stadtman, T. C. (1996). Properties of the selenium- and molybdenum-containing nicotinic acid hydroxylase from Clostridium barkeri. Biochemistry, 35(1), 212-23.
Gladyshev VN, Khangulov SV, Stadtman TC. Properties of the Selenium- and Molybdenum-containing Nicotinic Acid Hydroxylase From Clostridium Barkeri. Biochemistry. 1996 Jan 9;35(1):212-23. PubMed PMID: 8555176.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Properties of the selenium- and molybdenum-containing nicotinic acid hydroxylase from Clostridium barkeri. AU - Gladyshev,V N, AU - Khangulov,S V, AU - Stadtman,T C, PY - 1996/1/9/pubmed PY - 1996/1/9/medline PY - 1996/1/9/entrez SP - 212 EP - 23 JF - Biochemistry JO - Biochemistry VL - 35 IS - 1 N2 - NADP(+)-coupled nicotinic acid hydroxylase (NAH) has been purified to near-homogeneity from Clostridium barkeri by an improved purification scheme that allowed the isolation of milligram amounts of enzyme of higher specific activity then previously reported. NAH is most stable at alkaline pH in the presence of glycerol. The protein which consists of four dissimilar subunits occurs in forms of different molecular masses. There are 5-7 Fe, 1 FAD, and 1 Mo per 160 kDa protein promoter. Mo in the enzyme is bound to a dinucleotide form of molybdopterin and is coordinated with selenium. Mo(V), flavin radical, and two Fe2S2 clusters could be observed with EPR spectroscopy. The Se cofactor which is essential for nicotinic acid hydroxylase activity could be released from NAH as a reactive low molecular weight compound by a number of denaturing procedures. Parallel losses of Se and catalytic activity were observed during purification and storage of the enzyme. Addition of sodium selenide or selenophosphate did not restore the catalytic activity of the enzyme. Instead, NAH is reversibly inactivated by these compounds and also by sulfide. Cyanide, a common inhibitor of Mo-containing hydroxylases, does not affect NAH catalytic activity. The "as isolated" enzyme exhibits a Mo(V) EPR signal (2.067 signal) that was detected at early stages of purification. NAH exhibits a high substrate specificity toward electron donor substrates. The ability of a nicotinate analog to reduce NAH (disappearance of 2.067 signal) correlates with the rate of oxidation of the analog in the standard assay mixture. The properties of NAH differentiate the enzyme from known Mo-containing hydroxylases. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/8555176/Properties_of_the_selenium__and_molybdenum_containing_nicotinic_acid_hydroxylase_from_Clostridium_barkeri_ L2 - https://doi.org/10.1021/bi951793i DB - PRIME DP - Unbound Medicine ER -