20] Nicotinamide deamidase from Flavobacterium peregrinum

Abstract

This chapter discusses the formation of nicotinamide deamidase from Flavobacterium peregrinum . The first step in the biosynthesis of nicotinamide adenine dinucleotide (NAD) from nicotinamide in most microorganisms involves the deamidation of nicotinamide to form nicotinic acid and ammonia. This enzyme activity can be assayed by measuring ammonia formed colorimetrically or by measuring nicotinic acid formed or nicotinamide hydrolyzed. The standard deamidase reaction mixture contains 0.2 ml each of [7- 14 C]nicotinamide, maleate buffer, and MnCl 2 , plus deamidase to a total volume of 1.0 ml. After incubation for 15 min at 37°, the reaction is stopped by the addition of 0.5 ml of a 0.1 N acetic acid solution and heated in boiling water for 1 min. A 1.0-ml portion of the deproteinized reaction mixture is placed on a Dowex 1 X8 formate column. After the column has been washed, with 50 ml of water, the [7- 14 C]nicotinic acid is eluted, with 9 ml of 0.1 N formic acid, and the eluate is adjusted to 10 ml with water. The radioactivity recovered in this eluate is determined with a liquid scintillation spectrometer. Nicotinamide deamidase is not affected by MgCl 2 and CaCl 2 , whereas HgCl 2 , p -chloromercuribenzoate, ZnCl 2 , CuSO 4 , and FeCl 2 markedly inhibit enzyme activity. Enzyme activity is also inhibited by EDTA but not by α,α′-dipyridyl and o -phenanthroline.