Sequencing and heterologous expression of an epimerase and two lyases from iminodisuccinate-degrading bacteria

Abstract

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.

FIG. 1.

Extracts of a CLUSTAL_X (25) alignment of duck δII-crystallin (P24058), E. coli argininosuccinate lyase (AAC76942), and the amino acid sequences AAZ80811 (BY6-lyase) and AAZ80812 (SLRS7-lyase). Three domains with high sequence similarities within the fumarase II enzyme family (6, 22, 26, 27) are boxed. Positions of δII-crystallin amino acid residues involved in binding of argininosuccinate (21) are marked with arrowheads. The figure was prepared using the program GeneDoc (http://www.psc.edu/biomed/genedoc).