L-cysteate sulpho-lyase, a widespread pyridoxal 5'-phosphate-coupled desulphonative enzyme purified from Silicibacter pomeroyi DSS-3(T)

Abstract

Quantitative utilization of L-cysteate (2-amino-3-sulphopropionate) as the sole source of carbon and energy for growth of the aerobic, marine bacterium Silicibacter pomeroyi DSS-3(T) was observed. The sulphonate moiety was recovered in the medium largely as sulphite, and the appropriate amount of the ammonium ion was also observed. Genes [suyAB (3-sulpholactate sulpho-lyase)] encoding the known desulphonation reaction in cysteate degradation were absent from the genome, but a homologue of a putative sulphate exporter gene (suyZ) was found, and its neighbour, annotated as a D-cysteine desulphhydrase, was postulated to encode pyridoxal 5'-phosphate-coupled L-cysteate sulpho-lyase (CuyA), a novel enzyme. Inducible CuyA was detected in cysteate-grown cells. The enzyme released equimolar pyruvate, sulphite and the ammonium ion from L-cysteate and was purified to homogeneity by anion-exchange, hydrophobic-interaction and gel-filtration chromatography. The N-terminal amino acid sequence of this 39-kDa subunit confirmed the identification of the cuyA gene. The native enzyme was soluble and homomultimeric. The K(m)-value for L-cysteate was high (11.7 mM) and the enzyme also catalysed the D-cysteine desulphhydrase reaction. The gene cuyZ, encoding the putative sulphite exporter, was co-transcribed with cuyA. Sulphite was exported despite the presence of a ferricyanide-coupled sulphite dehydrogenase. CuyA was found in many bacteria that utilize cysteate.

Figure 1. CuyA, sulphite dehydrogenase and the transport processes presumed to be involved in the catabolism of L-cysteate by S. pomeroyi DSS-3 (A). The cuyAZ genes are SPOA0157 and SPOA0158; putative cuyR is SPOA0159 and the scale is in kbp (B)

Arrows in (B) indicate the positions of PCR primers. No information is available on the natures of sulphite dehydrogenase, the cysteate transport or ammonium export systems.

Figure 2. Growth of S. pomeroyi DSS-3 in 10 mM L-cysteate SBM-M (A) and the changes in concentrations of substrates and products during growth (B)

Key: ●, protein; ▲, cysteate; ■, sulphite; ○, ammonium ion.

Figure 3. Electropherograms of denatured proteins in extracts and fractions of S. pomeroyi DSS-3

Molecular mass standards are shown on the left hand side. Lane 1, extract of acetate-grown cells; lane 2, soluble fraction of L-cysteate-grown cells; lane 3, membrane fraction of cysteate-grown cells; lane 4, CuyA in eluate from the anion exchange column; lane 5, CuyA in eluate from the hydrophobic interaction column; lane 6, CuyA in eluate from the gel filtration column.

Figure 4. Pyruvate, sulphite and ammonium ions released from L-cysteate during the reaction of L-cysteate sulpho-lyase from S. pomeroyi DSS-3

Key: ■, sulphite; ○, ammonia; △, pyruvate; □ sulphite in the negative control (boiled extract or native extract of acetate-grown cells). Further negative controls confirmed that neither ammonium ion nor pyruvate was produced (results not shown).

Figure 5. Transcription of cuyZ during growth of S. pomeroyi DSS-3 with L-cysteate

Amplicons from a representative RT-PCR experiment were separated by gel electrophoresis; the predicted length of the amplicon from primer pair cuyZF/cuyZR was 200 bp. Lanes 1 and 6, 50 bp DNA ladder; lane 2, RT-PCR products from RNA of cysteate-grown cells of S. pomeroyi; lane 3, RT-PCR products from RNA of taurine-grown cells of S. pomeroyi; lane 4, negative control (no RNA); lane 5, positive control (S. pomeroyi DSS-3 chromosomal DNA as template).