Purification and biochemical characterization of some of the properties of recombinant human kynureninase


  • Enzyme: kynureninase (l-kynurenine hydrolase, EC

N. P. Botting, School of Chemistry, University of St Andrews, St Andrews, Fife KY16 9ST, Scotland, UK. Fax: + 44 1334 463808, Tel.: + 44 1334 463856, E-mail: npb@st-andrews.ac.uk


Recombinant human kynureninase (l-kynurenine hydrolase, EC was purified to homogeneity (60-fold) from Spodoptera frugiperda (Sf9) cells infected with baculovirus containing the kynureninase gene. The purification protocol comprised ammonium sulfate precipitation and several chromatographic steps, including DEAE–Sepharose CL-6B, hydroxyapatite, strong anionic and cationic separations. The purity of the enzyme was determined by SDS/PAGE, and the molecular mass verified by MALDI-TOF MS. The monomeric molecular mass of 52.4 kDa determined was > 99.99% of the predicted molecular mass. A UV absorption spectrum of the holoenzyme resulted in a peak at 432 nm. The optimum pH was 8.25 and the enzyme displayed a strong dependence on the ionic strength of the buffer for optimum activity. This cloned enzyme was highly specific for 3-hydroxykynurenine (Km = 3.0 µm ± 0.10) and was inhibited by l-kynurenine (Ki = 20 µm), d-kynurenine (Ki = 12 µm) and a synthetic substrate analogue d,l-3,7-dihydroxydesaminokynurenine (Ki = 100 nm). The activity/concentration profile for kynureninase from this source was sigmoidal in all instances. There appeared to be partial inhibition by substrate, and excess pyridoxal 5′-phosphate was found to be inhibitory.