The gene ENOD40 is expressed at an early stage of root nodule organogenesis and has been postulated to play a central regulatory role in the Rhizobium-legume interaction. In vitro translation of soybean ENOD40 mRNA showed that the gene encodes two peptides of 12 and 24 as residues (peptides A and B) that bind to sucrose synthase. Here we show that the small Cys-containing peptide A binds to sucrose synthase by disulfide bond formation, which may represent a novel form of posttranslational modification of this important metabolic enzyme. Assays using nanomolar concentrations of peptide A revealed that the monomeric reduced form of this peptide binds to purified sucrose synthase. Using a cysteinyl capture strategy combined with MALDI-TOF MS analysis we identified the Cys residue C264 of soybean sucrose synthase as the binding site of peptide A. Modification of sucrose synthase with ENOD40 peptide A activates sucrose cleavage activity whereas the synthesis activity of the enzyme is unaffected. The results are discussed in relation to the role of sucrose synthase in the control of sucrose utilization in nitrogen-fixing nodules. (C) 2004 Elsevier Inc. All rights reserved.