) -EJB 930930/1 P-protein, H-protein and T-protein of the glycine cleavage system have been purified from Escherichia coli. Their N-terminal amino acid sequences were determined, and a set of oligonucleotide probes was designed for gene cloning. The nucleotide sequence of a fragment of DNA around the 62-min region of the E. coli chromosome, containing genes for the components of the glycinecleavage system has been determined. The sequence i?cludes three structural genes encoding Tprotein (363 amino acids, 40013 Da), H-protein (128 amino acids, 13679 Da) and P-protein (956 amino acids, 104240Da). These genes are named gcvT, gcvH and gcvP, respectively. They are organized in the above-mentioned order on the same strand of DNA with short intercistronic sequences. The presence of a potential promoter preceding gcvT and a typical rho-independent terminator sequence following gcvP indicated that the three genes constitute a single operon. Each component of the E. coli glycine-cleavage system exhibits considerable amino acid sequence similarity with the animal and plant counterparts. When the plasmid containing the gcv operon was transfected in E. coli cells, the gene products of gcvT gcvH and gcvP were overexpressed under the direction of the promoter of the gcv operon. However, bacteria harboring the plasmid that contained the gcv operon without the promoter region and the 5' terminal portion of gcvT failed to overexpress any of the three components.The glycine-cleavage system is a niultienzyme complex that catalyzes the reversible oxidation of glycine, yielding carbon dioxide, ammonia, methylenetetrahydrofolate and a reduced pyridine nucleotide [l -51. The enzyme system has been purified from a number of sources including animal liver [6], plants [7,8] and bacteria such as Pepfococcus glycinophilus [9], Arthrobacter globformis [lo] and Eubacterium acidaminophilum [ll]. The system is composed of four proteins named p-, H-, T-and L-protein In Escherichia coli the glycine-cleavage system is inducible by exogenous glycine [24] and encoded by the gcv structural genes situated at 62 min in the linkage map [25]. Although a gene for H-protein has been sequenced [26], the detailed properties of the glycine-cleavage system in E. coli have not been examined so far. Our purpose is to characterize the structural genes for the components of the glycine-cleavage system from E. coli and to acquire more information about the characteristic features of the deduced amino acid sequences and the binding sites for coenzymes and prosthetic groups. In this study, we report the molecular cloning of these genes, their nucleotide sequences and deduced amino acid sequences and evidence for their arrangement in an operon. Where possible, these proteins have been compared to their animal and plant counterparts.