Purification, crystallization and preliminary crystallographic study of a recombinant plant aminoaldehyde dehydrogenase from Pisum sativum

Abstract

Aminoaldehydes are products of polyamine degradation and are known to be reactive metabolites that are toxic to living cells at high concentrations. These compounds are catabolized by aminoaldehyde dehydrogenases, which are enzymes that contain a nicotinamide adenine dinucleotide coenzyme. Aminoaldehyde dehydrogenase from Pisum sativum was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop method. A complete data set was collected to 2.8 A resolution at 100 K. Crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 86.4, b = 216.6, c = 205.4 A, beta = 98.1 degrees. Molecular replacement was performed and led to the identification of six dimers per asymmetric unit.

Figure 1

(a) Nu-PAGE and Western blot of a recombinant pea aminoaldehyde dehydrogenase (AMADH1). Nu-PAGE was performed using 4–12% bis-tris gel in MOPS buffer. The PVDF membrane was probed with polyclonal anti-AMADH rabbit antibodies, reacted with goat anti-rabbit IgG alkaline phosphatase conjugate and coloured using NBT/BCIP (nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate, Roche). (b) Crystals of AMADH1. The crystal shown (maximum dimension 0.3 mm) is similar to that used for the X-ray diffraction data collection.