Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus

Abstract

Proline dehydrogenase (PRODH) and ⌬ 1 -pyrroline-5-carboxylate dehydrogenase (P5CDH) catalyze the two-step oxidation of proline to glutamate. They are distinct monofunctional enzymes in all eukaryotes and some bacteria but are fused into bifunctional enzymes known as proline utilization A (PutA) in other bacteria. Here we report the first structure and biochemical data for a monofunctional PRODH. The 2.0-Å resolution structure of Thermus thermophilus PRODH reveals a distorted (␤␣) 8 barrel catalytic core domain and a hydrophobic ␣-helical domain located above the carboxylterminal ends of the strands of the barrel. Although the catalytic core is similar to that of the PutA PRODH domain, the FAD conformation of T. thermophilus PRODH is remarkably different and likely reflects unique requirements for membrane association and communication with P5CDH. Also, the FAD of T. thermophilus PRODH is highly solvent-exposed compared with PutA due to a 4-Å shift of helix 8. Structurebased sequence analysis of the PutA/PRODH family led us to identify nine conserved motifs involved in cofactor and substrate recognition. Biochemical studies show that the midpoint potential of the FAD is ؊75 mV and the kinetic parameters for proline are K m ‫؍‬ 27 mM and k cat ‫؍‬ 13 s ؊1 .3,4-Dehydro-L-proline was found to be an efficient substrate, and L-tetrahydro-2-furoic acid is a competitive inhibitor (K I ‫؍‬ 1.0 mM). Finally, we demonstrate that T. thermophilus PRODH reacts with O 2 producing superoxide. This is significant because superoxide production underlies the role of human PRODH in p53-mediated apoptosis, implying commonalities between eukaryotic and bacterial monofunctional PRODHs.Oxidation of amino acids is a central part of energy metabolism. The oxidative pathway for proline consists of two enzymatic steps and an intervening nonenzymatic equilibrium (Scheme 1) (1, 2). The first enzymatic step transforms proline to ⌬ 1 -pyrroline-5-carboxylate (P5C), 2 which is non-enzymatically hydrolyzed to glutamic semialdehyde. The semialdehyde is oxidized in the second enzymatic step to glutamate.This 4-electron transformation of proline is common to all organisms, but the enzymes of proline catabolism differ widely among the three kingdoms of life. Amino acid sequence analysis shows that bacteria and eukaryotes share a common set of proline catabolic enzymes called proline dehydrogenase (PRODH) and P5C dehydrogenase (P5CDH). Studies of the bacterial enzymes have shown that PRODH is an FAD-dependent enzyme with a (␤␣) 8 barrel catalytic core (3, 4), and P5CDH is an NAD ϩ -dependent Rossmann fold enzyme featuring a nucleophilic Cys (5). These enzymes are unrelated in sequence and structure to hyperthermophilic archaeal proline catabolic enzymes, which appear in unique hetero-tetrameric and hetero-octameric complexes (6).An intriguing aspect of proline catabolism in eukaryotes and bacteria is that PRODH and P5CDH are separate enzymes in some organisms, whereas the two enzymes are fused in other organisms. The traditional view has been that P...

Proline modulates the intracellular redox environment and protects mammalian cells against oxidative stress

“…6B, inset) [44]. These results are consistent with a previous study of PRODH from Thermus thermophilus which was also shown to generate superoxide during catalytic turnover and is thought to be an important model for understanding human PRODH [45]. To our knowledge, this is the first characterization of purified human PRODH and the in vitro assays demonstrate that superoxide is generated directly during catalytic turnover with proline.…”

Proline Utilization by Bacillus subtilis: Uptake and Catabolism

“…Database searches suggest that the B. subtilis PutB protein (303 amino acids) is a monofunctional PRODH (EC 1.5.99.8) (68). PutB exhibits 25% amino acid sequence identity to the PRODH domain (from amino acid 261 to amino acid 612) of the PutA enzyme from E. coli (61).…”

“…1A). We avoided the use of putA as a gene designation because the PutA protein in enterobacteria comprises both proline dehydrogenase (PRODH) and ⌬ 1 -pyrroline-5-carboxylate dehydrogenase (P5CDH) enzyme activities (37,52,59), whereas the PutB and PutC proteins from B. subtilis represent monofunctional PRODH and P5CDH enzymes (see below) (32,68). The putBCP gene cluster is followed in the same transcriptional orientation by the putR (ycgP) gene, which encodes the PutR protein, the proline-responsive activator of putBCP expression (7,31).…”

“…1B). These enzymatic steps can either be carried out by a single bifunctional protein comprising two domains (PRODH-P5CDH) (59) or by monofunctional proteins with separate PRODH and P5CDH activities (32,68).…”

Proline Dehydrogenase Contributes to Pathogen Defense in Arabidopsis

“…Remarkably, ProDH seems to promote mitochondrial oxidative burst in animal cells, where the POX gene is a target of p53 (Polyak et al, 1997), with POX overexpression triggering apoptosis by the accumulation of ROS at the mitochondria (Donald et al, 2001;Maxwell and Rivera, 2003). It is interesting that in Thermus thermophilus, ProDH autogenously catalyzes the generation of superoxide in vitro, thus demonstrating its structural flexibility in controlling the exposure of FAD to the solvent (White et al, 2007). However, it is not known whether any of these features are conserved in plant ProDHs.…”