title: The differential ability of asparagine and glutamine in promoting the closed/active enzyme conformation rationalizes the Wolinella succinogenes L-asparaginase substrate specificity.
abstract: Many side effects of current FDA-approved L-asparaginases have been related to their secondary L-glutaminase activity. The Wolinella succinogenes L-asparaginase (WoA) has been reported to be L-glutaminase free, suggesting it would have fewer side effects. Unexpectedly, the WoA variant with a proline at position 121 (WoA-P121) was found to have L-glutaminase activity in contrast to Uniprot entry P50286 (WoA-S121) that has a serine residue at this position. Towards understanding how this residue impacts the L-glutaminase property, kinetic analysis was coupled with crystal structure determination of these WoA variants. WoA-S121was confirmed to have much lower L-glutaminase activity than WoA-P121, yet both showed comparable L-asparaginase activity. Structures of the WoA variants in complex with L-aspartic acid versus L-glutamic acid provide insights into their differential substrate selectivity. Structural analysis suggests a mechanism by which residue 121 impacts the conformation of the conserved tyrosine 27, a component of the catalytically-important flexible N-terminal loop. Surprisingly, we could fully model this loop in either its open or closed conformations, revealing the roles of specific residues of an evolutionary conserved motif among this L-asparaginase family. Together, this work showcases critical residues that influence the ability of the flexible N-terminal loop for adopting its active conformation, thereby effecting substrate specificity.
authors.affiliation: The Jesse Brown VA Medical Center, Chicago, Illinois, United States of America.|Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois, United States of America.
authors.forename: Hien Anh
authors.initials: HA
authors.lastname: Nguyen
authors.affiliation: Department of Pediatrics, Division of Pediatric Hematology-Oncology, Moores Cancer Center, University of California San Diego Health System, La Jolla, California, United States of America.
authors.forename: Donald L
authors.initials: DL
authors.lastname: Durden
authors.affiliation: The Jesse Brown VA Medical Center, Chicago, Illinois, United States of America.|Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois, United States of America.
authors.forename: Arnon
authors.initials: A
authors.lastname: Lavie
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D001215:Asparaginase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001216:Asparagine / Q000737:chemistry / Q000378:metabolism*; D017124:Conserved Sequence; D004789:Enzyme Activation; D005972:Glutaminase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D017354:Point Mutation; D011487:Protein Conformation; D013379:Substrate Specificity; D016968:Wolinella / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D005973:Glutamine; D001216:Asparagine; D001215:Asparaginase; D005972:Glutaminase