Sequencing and heterologous expression of an epimerase and two lyases from iminodisuccinate-degrading bacteria.
Recently, degradation of all existing epimers of the complexing agent iminodisuccinate (IDS) in the bacterial strain Agrobacterium tumefaciens BY6 was proven to depend on an epimerase and a C-N lyase (Cokesa et al., Appl. Environ. Microbiol. 70:3941-3947, 2004). In the bacterial strain Ralstonia sp. strain SLRS7, a corresponding C-N lyase is responsible for the initial degradation step (Cokesa et al., Biodegradation 15:229-239, 2004). The ite gene, encoding the IDS-transforming epimerase, and the genes icl(B) and icl(S), encoding the IDS-converting BY6-lyase and SLRS7-lyase, respectively, were cloned and sequenced. The epimerase gene encodes a protein with a predicted subunit molecular mass of 47.6 kDa. The highest degree of epimerase amino acid sequence identities was found with proteins of unknown function, indicating a novel protein. For the lyases, the deduced amino acid sequences show high similarity to enzymes of the fumarase II family. A classification into a new subfamily within the enzyme family is proposed. The subunit molecular masses of the lyases were calculated to be 54.4 and 54.7 kDa, respectively. In Agrobacterium tumefaciens BY6, the ite gene was on an approximately 180-kb circular plasmid, whereas the icl(B) gene was chromosomal like the corresponding icl(S) gene in Ralstonia sp. strain SLRS7. Heterologous expression in Escherichia coli and subsequent purification revealed recombinant enzymes with in vitro activity similar to that of the corresponding enzymes from the wild-type strains.
Bäuerle
Bettina
B
Institute of Microbiology, University of Stuttgart, 70569 Stuttgart, Germany.
Cokesa
Zeljko
Z
Hofmann
Silvia
S
Rieger
Paul-Gerhard
PG
D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008190:Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D019998:Racemases and Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043367:Ralstonia / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013386:Succinates / Q000378:metabolism*
D000596:Amino Acids; D013386:Succinates; C412825:iminodisuccinic acid; D008190:Lyases; D019998:Racemases and Epimerases