Structure, function, and mechanism of proline utilization A (PutA)

Abstract

Proline has important roles in multiple biological processes such as cellular bioenergetics, cell growth, oxidative and osmotic stress response, protein folding and stability, and redox signaling. The proline catabolic pathway, which forms glutamate, enables organisms to utilize proline as a carbon, nitrogen, and energy source. FAD-dependent proline dehydrogenase (PRODH) and NAD+-dependent glutamate semialdehyde dehydrogenase (GSALDH) convert proline to glutamate in two sequential oxidative steps. Depletion of PRODH and GSALDH in humans leads to hyperprolinemia, which is associated with mental disorders such as schizophrenia. Also, some pathogens require proline catabolism for virulence. A unique aspect of proline catabolism is the multifunctional proline utilization A (PutA) enzyme found in Gram-negative bacteria. PutA is a large (> 1000 residues) bifunctional enzyme that combines PRODH and GSALDH activities into one polypeptide chain. In addition, some PutAs function as a DNA-binding transcriptional repressor of proline utilization genes. This review describes several attributes of PutA that make it a remarkable flavoenzyme: (1) diversity of oligomeric state and quaternary structure; (2) substrate channeling and enzyme hysteresis; (3) DNA-binding activity and transcriptional repressor function; and (4) flavin redox dependent changes in subcellular location and function in response to proline (functional switching).

Discovery of the Membrane Binding Domain in Trifunctional Proline Utilization A

“…1–4 Cellular energy is generated from the oxidative breakdown of proline to glutamate via the enzymes proline dehydrogenase (PRODH) and L-glutamate- γ -semialdehyde dehydrogenase (GSALDH), which in eukaryotes are localized in the mitochondrion. 2,5,6 In humans, PRODH has been shown to influence cancer development and progression, with suppressor and pro-metastasis functions in early and late stage cancer, respectively. 2,4,7–10 Inborn deficiencies in PRODH are associated with hyperprolineamia type 1, 11 risk of schizophrenia, 11–13 and autism spectrum disorder.…”

“…5,23 PutA enzymes are unique to Gram-negative bacteria, as eukaryotes and Gram-positive bacteria encode separate PRODH and GSALDH enzymes. 5 The oxidation of proline to glutamate is initiated by PRODH, which generates Δ 1 -pyrroline-5-carboxylate (P5C) (Figure 1A).…”

“…5,23 PutA enzymes are unique to Gram-negative bacteria, as eukaryotes and Gram-positive bacteria encode separate PRODH and GSALDH enzymes. 5 The oxidation of proline to glutamate is initiated by PRODH, which generates Δ 1 -pyrroline-5-carboxylate (P5C) (Figure 1A). PRODH is a FAD-dependent ( βα ) 8 barrel domain enzyme that couples the oxidation of proline to reduction of ubiquinone in the respiratory chain.…”

“…PRODH is a FAD-dependent ( βα ) 8 barrel domain enzyme that couples the oxidation of proline to reduction of ubiquinone in the respiratory chain. 5,24–26 GSALDH (aka P5C dehydrogenase and ALDH4A1) catalyzes the NAD + -dependent oxidation of L-glutamate- γ -semialdehyde (GSAL), which is generated in an intervening nonenzymatic step by hydrolysis of P5C. 5,27 A substrate channel links the PRODH and GSALDH domains in PutAs, thereby preventing P5C/GSAL loss to the cellular milieu and improving efficiency of glutamate production from pro-line.…”

“…5,36 Type A PutAs, which lack the ALDHSF domain, have a different oligomeric structure than type B PutAs and form the substrate channel via domain-swapped dimerization. 5 Finally, a group of PutA proteins classified as type C contain a N-terminal DNA-binding domain and are known as “trifunctional PutAs” (Figure 1B). 5 The DNA-binding domain has a ribbon-helix-helix (RHH) fold and has been structurally characterized from Escherichia coli 37,38 and Pseudomonas putida.…”