Structure of the proline dehydrogenase domain of the multifunctional PutA flavoprotein

Abstract

The PutA flavoprotein from Escherichia coli plays multiple roles in proline catabolism by functioning as a membrane-associated bi-functional enzyme and a transcriptional repressor of proline utilization genes. The human homolog of the PutA proline dehydrogenase (PRODH) domain is critical in p53-mediated apoptosis and schizophrenia. Here we report the crystal structure of a 669-residue truncated form of PutA that shows both PRODH and DNA-binding activities, representing the first structure of a PutA protein and a PRODH enzyme from any organism. The structure is a domain-swapped dimer with each subunit comprising three domains: a helical dimerization arm, a 120-residue domain containing a three-helix bundle similar to that in the helix-turn-helix superfamily of DNA-binding proteins and a beta/alpha-barrel PRODH domain with a bound lactate inhibitor. Analysis of the structure provides insight into the mechanism of proline oxidation to pyrroline-5-carboxylate, and functional studies of a mutant protein suggest that the DNA-binding domain is located within the N-terminal 261 residues of E. coli PutA.

PrcR, a PucR-type transcriptional activator, is essential for proline utilization and mediates proline-responsive expression of the proline utilization operon putBCP in Bacillus subtilis

“…In Escherichia coli, Salmonella typhimurium and some other bacteria, the putA gene encodes a bifunctional proline utilization protein with both the proline dehydrogenase and D 1 -pyrroline-5-carboxylate dehydrogenase activities for the two-step oxidation of proline to glutamate (Menzel & Roth, 1981;Lee et al, 2003;Tanner, 2008). In some of these bacteria, the PutA protein can also act as a transcriptional repressor for controlling expression of the putA and putP genes.…”

“…Exogenous proline can induce the expression of the putBCP operon and proline dehydrogenase activity in B. subtilis (Atkinson et al, 1990;Bremer, 2002). However, the regulatory mechanism for proline induction of putBCP expression in B. subtilis remains unknown.In Escherichia coli, Salmonella typhimurium and some other bacteria, the putA gene encodes a bifunctional proline utilization protein with both the proline dehydrogenase and D 1 -pyrroline-5-carboxylate dehydrogenase activities for the two-step oxidation of proline to glutamate (Menzel & Roth, 1981;Lee et al, 2003;Tanner, 2008). In some of these bacteria, the PutA protein can also act as a transcriptional repressor for controlling expression of the putA and putP genes.…”

Identification and Characterization of the DNA-binding Domain of the Multifunctional PutA Flavoenzyme

“…The recognition helix (residues 230 -241) of the HTH motif from the PutA669 structure contains basic residues that seem properly positioned for ionic and hydrogen bond interactions with DNA such as Arg-230, Arg-234, and Lys-238. PutA261, a truncated PutA protein containing resi-dues 1-261, was shown to bind the put intergenic DNA region, demonstrating that DNA-binding activity can be separated from the PRODH domain and further implying that residues 139 -258 were involved in DNA binding (13).…”

“…A truncated form of PutA containing residues 1-669 (PutA669) was shown to exhibit proline dehydrogenase (PRODH) and DNA binding activities but lack P5C dehydrogenase activity (12). The x-ray crystal structure of PutA669 complexed to the competitive inhibitor L-lactate was solved to 2.0-Å resolution (13). The crystal structure included residues 87-612 of PutA669 and revealed that the PRODH domain is a ␤ 8 ␣ 8 barrel composed of residues 261-612, with the FAD bound at the C-terminal ends of the ␤-strands of the barrel.…”

Expression, Purification and Characterization of the Proline Dehydrogenase Domain of PutA from Pseudomonas putida POS-F84

“…Multifunctional PutA flavoenzyme contains ProDH and P5CDH domains [3]. In addition to these enzymatic roles, PutA polypeptide has also DNA-binding activity and participates in the transcriptional control of put genes [4]. In the absence of proline, PutA accumulates in the cytoplasm and represses transcription of the put regulon by binding to the control intergenic region between putP and putA genes.…”