Crystallization and preliminary crystallographic analysis of the proline dehydrogenase domain of the multifunctional PutA flavoprotein fromEscherichia coli

Abstract

The PutA flavoprotein from Escherichia coli is a multifunctional protein that plays pivotal roles in proline catabolism by functioning as both a membrane‐associated bifunctional enzyme and a transcriptional repressor. Peripherally membrane‐bound PutA catalyzes the two‐step oxidation of proline to glutamate, while cytoplasmic PutA represses the transcription of its own gene and the gene for a proline‐transporter protein. X‐ray crystallographic studies on PutA have been initiated to determine how the PutA structural scaffold enables it to be both an enzyme and a repressor, and to understand the mechanism by which PutA switches between its enzymatic and DNA‐­binding functions. To facilitate crystallization, a recombinant protein (PutA669) corresponding to the N‐terminal 669 amino‐acid residues of the 1320 residues of PutA was engineered. Activity assays demonstrated that PutA669 catalyzes the first step of chemistry performed by PutA, the conversion of proline to Δ1‐pyrroline‐5‐­carboxylate. Crystals of PutA669 have been obtained from PEG 3000 buffered at pH 6–7. The crystals occupy an I‐centered orthorhombic lattice with unit‐cell parameters a = 72.5, b = 140.2, c = 146.8 Å; a 2.15 Å data set was collected using a rotating‐anode source. Assuming one molecule per asymmetric unit, the Matthews coefficient VM is 2.5 Å3 Da−1, with a solvent content of 50%. The structure of PutA669 will be solved by multiple isomorphous replacement.

Structures of the Escherichia coli PutA Proline Dehydrogenase Domain in Complex with Competitive Inhibitors,

“…PutA86-669 was prepared in pUTA669 [a pET-23b construct encoding residues 1-669 of PutA with a C-terminal hexahistidine tag (29)] using QuikChange (Stratagene) sitedirected mutagenesis. An NdeI site was introduced at amino acid codon 84 of the putA gene in pUTA669.…”

Characterization of a Bifunctional PutA Homologue fromBradyrhizobium japonicumand Identification of an Active Site Residue that Modulates Proline Reduction of the Flavin Adenine Dinucleotide Cofactor

“…Wild-type BjPutA and mutant A310V were overexpressed as N-terminal 8xHis tag fusion proteins from pKA8H-BjPutA in E. coli strain BL21 DE3 pLysS and purified using a Ni 2+ NTA affinity column (Novagen) and anion exchange chromatography (Hi-Trap™ Qsepharose, Amersham Biosciences) as previously described for EcPutA (25)(26)(27). The Nterminal 8xHis tag was retained after purification.…”

“…The resulting construct, pKA8H-BjPutA, was verified by nucleic acid sequencing. The BjPutA mutant A310V was engineered by QuikChange site-directed mutagenesis using primers 5′ -GACGGTTTTGGGCTCGTGATCCAGGCCTATCAG -3′ and 5′ -CTGATAGGCCTGGATCACGAGCCCAAAACCGTC -3'.Wild-type BjPutA and mutant A310V were overexpressed as N-terminal 8xHis tag fusion proteins from pKA8H-BjPutA in E. coli strain BL21 DE3 pLysS and purified using a Ni 2+ NTA affinity column (Novagen) and anion exchange chromatography (Hi-Trap™ Qsepharose, Amersham Biosciences) as previously described for EcPutA (25)(26)(27). The Nterminal 8xHis tag was retained after purification.…”

Cloning, purification and crystallization ofThermus thermophilusproline dehydrogenase

“…In addition, the crystal structure of the PRODH domain of E. coli PutA has been determined (Nadaraia et al, 2001;Lee et al, 2003;Zhang, White et al, 2004). The structures of PutA P5CDH and DNA-binding domains are currently not known.…”