title: Structure determination and biochemical studies on Bacillus stearothermophilus E53Q serine hydroxymethyltransferase and its complexes provide insights on function and enzyme memory.
abstract: Serine hydroxymethyltransferase (SHMT) belongs to the alpha-family of pyridoxal 5'-phosphate-dependent enzymes and catalyzes the reversible conversion of L-Ser and tetrahydrofolate to Gly and 5,10-methylene tetrahydrofolate. 5,10-Methylene tetrahydrofolate serves as a source of one-carbon fragment in many biological processes. SHMT also catalyzes the tetrahydrofolate-independent conversion of L-allo-Thr to Gly and acetaldehyde. The crystal structure of Bacillus stearothermophilus SHMT (bsSHMT) suggested that E53 interacts with the substrate, L-Ser and tetrahydrofolate. To elucidate the role of E53, it was mutated to Q and structural and biochemical studies were carried out with the mutant enzyme. The internal aldimine structure of E53QbsSHMT was similar to that of the wild-type enzyme, except for significant changes at Q53, Y60 and Y61. The carboxyl of Gly and side chain of L-Ser were in two conformations in the respective external aldimine structures. The mutant enzyme was completely inactive for tetrahydrofolate-dependent cleavage of L-Ser, whereas there was a 1.5-fold increase in the rate of tetrahydrofolate-independent reaction with L-allo-Thr. The results obtained from these studies suggest that E53 plays an essential role in tetrahydrofolate/5-formyl tetrahydrofolate binding and in the proper positioning of Cbeta of L-Ser for direct attack by N5 of tetrahydrofolate. Most interestingly, the structure of the complex obtained by cocrystallization of E53QbsSHMT with Gly and 5-formyl tetrahydrofolate revealed the gem-diamine form of pyridoxal 5'-phosphate bound to Gly and active site Lys. However, density for 5-formyl tetrahydrofolate was not observed. Gly carboxylate was in a single conformation, whereas pyridoxal 5'-phosphate had two distinct conformations. The differences between the structures of this complex and Gly external aldimine suggest that the changes induced by initial binding of 5-formyl tetrahydrofolate are retained even though 5-formyl tetrahydrofolate is absent in the final structure. Spectral studies carried out with this mutant enzyme also suggest that 5-formyl tetrahydrofolate binds to the E53QbsSHMT-Gly complex forming a quinonoid intermediate and falls off within 4 h of dialysis, leaving behind the mutant enzyme in the gem-diamine form. This is the first report to provide direct evidence for enzyme memory based on the crystal structure of enzyme complexes.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors.forename: V
authors.initials: V
authors.lastname: Rajaram
authors.forename: B S
authors.initials: BS
authors.lastname: Bhavani
authors.forename: Purnima
authors.initials: P
authors.lastname: Kaul
authors.forename: V
authors.initials: V
authors.lastname: Prakash
authors.forename: N
authors.initials: N
authors.lastname: Appaji Rao
authors.forename: H S
authors.initials: HS
authors.lastname: Savithri
authors.forename: M R N
authors.initials: MR
authors.lastname: Murthy
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D005575:Formyltetrahydrofolates / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D012696:Glycine Hydroxymethyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D013053:Spectrophotometry; D013763:Tetrahydrofolates / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005575:Formyltetrahydrofolates; D013763:Tetrahydrofolates; D005973:Glutamine; D018698:Glutamic Acid; C030371:5,6,7,8-tetrahydrofolic acid; D012696:Glycine Hydroxymethyltransferase; D005998:Glycine