A stereoselective carbon-nitrogen lyase from Ralstonia sp. SLRS7 cleaves two of three isomers of iminodisuccinate.
Following biodegradation tests according to the OECD guidelines for testing of chemicals 301F different degradation rates were observed for the three stereoisomers of iminodisuccinate (IDS). A strain was isolated from activated sludge, which used two of three isomers, R,S-IDS and S,S-IDS, as sole source of carbon, nitrogen, and energy. The isolated strain was identified by 16S-rDNA and referred to as Ralstonia sp. SLRS7. An IDS-degrading lyase was isolated from the cell-free extract. The enzyme was purified by three chromatographic steps, which included anion-exchange chromatography, hydrophobic interaction chromatography and gel filtration. The lyase catalysed the non-hydrolytic cleavage of IDS without requirement of any cofactors. Cleavage of S,S-IDS led to the formation of fumaric acid and L-aspartic acid. Interestingly R,S-IDS yielded only D-aspartic acid besides fumaric acid. R,R-IDS was not transformed. Thus, the IDS-degrading enzyme is a carbon-nitrogen lyase attacking only the asymmetric carbon atom exhibiting the S-configuration. Besides S,S-IDS and R,S-IDS cleavage, the lyase catalysed also the transformation of certain S,S-IDS metal complexes, namely Ca(2+)-, Mg(2+)- and Mn(2+)-IDS. The maximum enzyme activity was found at pH 8.0-8.5 and 35 degrees C. SDS-PAGE analysis revealed a single 57-kDa protein band. The native enzyme was estimated to be around 240 kDa indicating a homotetramer enzyme.
Institut für Mikrobiologie der Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
Zeljko
Z
Cokesa
Silvia
S
Lakner
Hans-Joachim
HJ
Knackmuss
Paul-Gerhard
PG
Rieger
D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D001673:Biodegradation, Environmental; D019759:Carbon-Nitrogen Lyases / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D004492:Edetic Acid / Q000737:chemistry; D005029:Ethylenediamines / Q000737:chemistry; D015394:Molecular Structure; D008970:Molecular Weight; D043367:Ralstonia / Q000201:enzymology / Q000302:isolation & purification; D012722:Sewage / Q000382:microbiology; D013237:Stereoisomerism; D013379:Substrate Specificity; D013386:Succinates / Q000737:chemistry* / Q000378:metabolism
C499100:(S,S)-ethylenediaminedisuccinate; D000596:Amino Acids; D003470:Culture Media; D005029:Ethylenediamines; D012722:Sewage; D013386:Succinates; C412825:iminodisuccinic acid; D004492:Edetic Acid; D019759:Carbon-Nitrogen Lyases