Pseudomonas putida Proline Utilization A DNA Binding Domain (PpPutA45)

Proline utilization A (PutA) is a multifunctional enzyme that allows Gram-negative bacteria, such as Escherichia coli and Pseudomonas putida, to utilize proline as a carbon and nitrogen source. PutA converts proline into glutamate in a two-step process that requires a proline dehydrogenase domain (PRODH) and a D¹-pyrroline-5-carboxylate (P5C) dehydrogenase domain (P5CDH). PRODH and P5CDH are separate enzymes in Gram-positive bacteria, archea, and eukaryotes. Proline is first oxidized to P5C coupled with the reduction of the FAD cofactor by the PutA PRODH domain. The reduced FADH₂ cofactor transfers the electrons to the electron transport chain system in the cytoplasmic membrane. The P5CDH domain then catalyzes the NAD⁺-dependant oxidation of P5C to glutamate.

(from J. J. Tanner Amino Acids (2008) 35:719-730)

PutA also functions as an autogenous transcriptional repressor by binding to multiple sites in the put regulatory region. Because the enzymatic activity of PutA requires PutA to be peripherally membrane bound, PutA function is regulated by proline-dependent switching of its intracellular location from the cytoplasm to the membrane. PutA can bind to the put control DNA in the absence (K_D ~45 nM) and presence (K_D~ 100 nM) of proline suggesting changes in PutA-DNA binding affinity are not a major factor in functional switching. Rather, proline reduction of the FAD cofactor activates tight PutA-lipid binding (K_D < 0.01nM) leading to sequestration of PutA on the membrane. The tight membrane interaction thus prevents PutA from binding DNA. Previous studies have shown redox-dependent conformational changes occur in a linker region between the DNA binding and PRODH domains. Thus, a coupled conformational change involving the PRODH and DNA binding domains may be part of the redox mechanism by which PutA transfers from the cytoplasm to the membrane. The PutA DNA binding domain in E. coli is localized to the N-terminal 47 amino acids and is separated from the PRODH domain (261-612) by a flexible domain of unknown function (residues 141-260). This flexible domain incurs a significant conformational change upon proline binding, where membrane association of PutA is primarily driven by a hydrophobic interaction.

P. putida is a non-pathogenic organism with utility in bioremediation because of its metabolic diversity and ability to metabolize a wide-range of carbon sources. PutA from P. putida is a 1315 amino acid polypeptide and functions as a transcriptional repressor of the put regulon in a manner similar to E. coli PutA. The N-terminal residues 1-45 of PutA from P. putida (PpPutA45), was shown to be responsible for DNA binding and dimerization. The protein is  a symmetrical homodimer (12 kDa) consisting of two ribbon-helix-helix (RHH) structures. PpPutA45 binds a 14 base-pair DNA oligomer (5’-GCGGTTGCACCTTT-3’). The antiparallel b-sheet that results from PpPutA45 dimerization serves as the DNA recognition binding site by inserting into the DNA major groove. The dimeric core of four α-helices provides a structural scaffold for the b-sheet from which residues Thr5, Gly7, and Lys9 make sequence specific contacts with the DNA. The structural model implies flexibility of Lys9 which can either make hydrogen bond contacts with guanine or thymine. The high sequence and structure conservation of the PutA RHH domain suggest interdomain interactions play an important role in the evolution of the protein.

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