4jny | pdb_00004jny
Crystal structure of PutA86-630 mutant D370A complexed with L-Tetrahydro-2-furoic acid
Structural highlights
Function[PUTA_ECOLI] Oxidizes proline to glutamate for use as a carbon and nitrogen source and also function as a transcriptional repressor of the put operon. Publication Abstract from PubMedProline utilization A (PutA) from Escherichia coli is a membrane-associated trifunctional flavoenzyme that catalyzes the oxidation of proline to glutamate and moonlights as a transcriptional regulator. As a regulatory protein, PutA represses transcription of the put regulon, which contains the genes encoding PutA and the proline transporter PutP. The binding of proline to the proline dehydrogenase active site and the subsequent reduction of the flavin induce high affinity membrane association of PutA and relieve repression of the put regulon, thereby causing PutA to switch from its regulatory to its enzymatic role. Here, we present evidence suggesting that residues of the beta3-alpha3 loop of the proline dehydrogenase domain (betaalpha)8 barrel are involved in proline-mediated allosteric regulation of PutA-membrane binding. Mutation of the conserved residues Asp370 and Glu372 in the beta3-alpha3 loop abrogates the ability of proline to induce functional membrane association. Both in vitro lipid/membrane binding assays and in vivo cell-based assays demonstrate that mutagenesis of Asp370 (D370N/A) or Glu372 (E372A) dramatically impedes PutA functional switching. The crystal structures of the proline dehydrogenase domain mutants PutA86-630D370N and PutA86-630D370A complexed with the proline analogue l-tetrahydro-2-furoic acid show that the mutations cause only minor perturbations to the active site but no major structural changes, suggesting that the lack of proline response is not due to a failure of the mutated active sites to correctly bind the substrate. Rather, these results suggest that the beta3-alpha3 loop may be involved in transmitting the status of the proline dehydrogenase active site and flavin redox state to the distal membrane association domain. Involvement of the beta3-alpha3 loop of the proline dehydrogenase domain in allosteric regulation of membrane association of proline utilization A.,Zhu W, Haile AM, Singh RK, Larson JD, Smithen D, Chan JY, Tanner JJ, Becker DF Biochemistry. 2013 Jul 2;52(26):4482-91. doi: 10.1021/bi400396g. Epub 2013 Jun, 19. PMID:23713611[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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