Cloning and sequencing of two Ceriporiopsis subvermispora bicupin oxalate oxidase allelic isoforms: implications for the reaction specificity of oxalate oxidases and decarboxylases.
Oxalate oxidase is thought to be involved in the production of hydrogen peroxide for lignin degradation by the dikaryotic white rot fungus Ceriporiopsis subvermispora. This enzyme was purified, and after digestion with trypsin, peptide fragments of the enzyme were sequenced using quadrupole time-of-flight mass spectrometry. Starting with degenerate primers based on the peptide sequences, two genes encoding isoforms of the enzyme were cloned, sequenced, and shown to be allelic. Both genes contained 14 introns. The sequences of the isoforms revealed that they were both bicupins that unexpectedly shared the greatest similarity to microbial bicupin oxalate decarboxylases rather than monocupin plant oxalate oxidases (also known as germins). We have shown that both fungal isoforms, one of which was heterologously expressed in Escherichia coli, are indeed oxalate oxidases that possess < or =0.2% oxalate decarboxylase activity and that the organism is capable of rapidly degrading exogenously supplied oxalate. They are therefore the first bicupin oxalate oxidases to have been described. Heterologous expression of active enzyme was dependent on the addition of manganese salts to the growth medium. Molecular modeling provides new and independent evidence for the identity of the catalytic site and the key amino acid involved in defining the reaction specificities of oxalate oxidases and oxalate decarboxylases.
Biological Chemistry Department, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom. Stephen.Bornemann@bbsrc.ac.uk.
Marta R
MR
Escutia
Laura
L
Bowater
Anne
A
Edwards
Andrew R
AR
Bottrill
Matthew R
MR
Burrell
Rubén
R
Polanco
Rafael
R
Vicuña
Stephen
S
Bornemann
D000483:Alleles; D000595:Amino Acid Sequence; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D007527:Isoenzymes; D008969:Molecular Sequence Data; D010070:Oxalates / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020072:Polyporales / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D007527:Isoenzymes; D010070:Oxalates; D010088:Oxidoreductases; C020598:oxalate oxidase; D002262:Carboxy-Lyases; C021915:oxalate decarboxylase