TY - JOUR
T1 - Cloning, expression, characterisation and mutational analysis of l-aspartate oxidase from Pseudomonas putida
AU - Leese, C.
AU - Grogan, G.
AU - Fotheringham, I.
AU - Escalettes, F.
AU - Speight, R.
PY - 2013/1/1
Y1 - 2013/1/1
N2 - l-Amino acid oxidases (LAAOs) are useful catalysts for the deracemisation of racemic amino acid substrates when combined with abiotic reductants. The gene nadB encoding the l-aspartate amino acid oxidase from Pseudomonas putida (PpLASPO) has been cloned and expressed in E. coli. The purified PpLASPO enzyme displayed a K for l-aspartic acid of 2.26 mM and a k = 10.6 s, with lower activity also displayed towards l-asparagine, for which pronounced substrate inhibition was also observed. The pH optimum of the enzyme was recorded at pH 7.4. The enzyme was stable for 60 min at up to 40 °C, but rapid losses in activity were observed at 50 °C. A mutational analysis of the enzyme, based on its sequence homology with the LASPO from E. coli of known structure, appeared to confirm roles in substrate binding or catalysis for residues His244, His351, Arg386 and Arg290 and also for Thr259 and Gln242. The high activity of the enzyme, and its promiscuous acceptance of both l-asparagine and l-glutamate as substrates, if with low activity, suggests that PpLASPO may provide a good model enzyme for evolution studies towards AAOs of altered or improved properties in the future.
AB - l-Amino acid oxidases (LAAOs) are useful catalysts for the deracemisation of racemic amino acid substrates when combined with abiotic reductants. The gene nadB encoding the l-aspartate amino acid oxidase from Pseudomonas putida (PpLASPO) has been cloned and expressed in E. coli. The purified PpLASPO enzyme displayed a K for l-aspartic acid of 2.26 mM and a k = 10.6 s, with lower activity also displayed towards l-asparagine, for which pronounced substrate inhibition was also observed. The pH optimum of the enzyme was recorded at pH 7.4. The enzyme was stable for 60 min at up to 40 °C, but rapid losses in activity were observed at 50 °C. A mutational analysis of the enzyme, based on its sequence homology with the LASPO from E. coli of known structure, appeared to confirm roles in substrate binding or catalysis for residues His244, His351, Arg386 and Arg290 and also for Thr259 and Gln242. The high activity of the enzyme, and its promiscuous acceptance of both l-asparagine and l-glutamate as substrates, if with low activity, suggests that PpLASPO may provide a good model enzyme for evolution studies towards AAOs of altered or improved properties in the future.
UR - http://www.scopus.com/inward/record.url?scp=84865650221&partnerID=8YFLogxK
U2 - 10.1016/j.molcatb.2012.07.008
DO - 10.1016/j.molcatb.2012.07.008
M3 - Article
AN - SCOPUS:84865650221
SN - 1381-1177
VL - 85-86
SP - 17
EP - 22
JO - Journal of Molecular Catalysis B : Enzymatic
JF - Journal of Molecular Catalysis B : Enzymatic
ER -