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

Abstract

Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the flavin-dependent oxidation of proline to Delta(1)-pyrroline-5-carboxylate. Here we present a structure-based study of the PRODH active site of the multifunctional Escherichia coli proline utilization A (PutA) protein using X-ray crystallography, enzyme kinetic measurements, and site-directed mutagenesis. Structures of the PutA PRODH domain complexed with competitive inhibitors acetate (K(i) = 30 mM), L-lactate (K(i) = 1 mM), and L-tetrahydro-2-furoic acid (L-THFA, K(i) = 0.2 mM) have been determined to high-resolution limits of 2.1-2.0 A. The discovery of acetate as a competitive inhibitor suggests that the carboxyl is the minimum functional group recognized by the active site, and the structures show how the enzyme exploits hydrogen-bonding and nonpolar interactions to optimize affinity for the substrate. The PRODH/L-THFA complex is the first structure of PRODH with a five-membered ring proline analogue bound in the active site and thus provides new insights into substrate recognition and the catalytic mechanism. The ring of L-THFA is nearly parallel to the middle ring of the FAD isoalloxazine, with the inhibitor C5 atom 3.3 A from the FAD N5. This geometry suggests direct hydride transfer as a plausible mechanism. Mutation of conserved active site residue Leu432 to Pro caused a 5-fold decrease in k(cat) and a severe loss in thermostability. These changes are consistent with the location of Leu432 in the hydrophobic core near residues that directly contact FAD. Our results suggest that the molecular basis for increased plasma proline levels in schizophrenic subjects carrying the missense mutation L441P is due to decreased stability of human PRODH2.

Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus

“…The TtPRODH structure is only the second PRODH structure solved to date, with the first being the PRODH domain of E. coli PutA (PutA86 -669, PDB code 1TIW) (3,4). Residues 263-561 of PutA form a distorted TIM barrel that is similar to that of TtPRODH ( Fig.…”

“…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).…”

Cloning, purification and crystallization ofThermus thermophilusproline dehydrogenase

“…The enzyme displayed PRODH activity as measured by a previously described assay (Zhang, White et al, 2004). The molecular mass as determined by MALDI-TOF mass spectrometry at the University of Missouri-Columbia Proteomics Center was 37968 AE 3.…”

“…Molecular-replacement calculations (high-resolution limit = 4 Å ) were performed with MOLREP (Vagin & Teplyakov, 2000) using the 8 8 barrel of the PRODH domain of E. coli PutA (Lee et al, 2003;Zhang, White et al, 2004) as the search model (residues 264-435 and 457-562 of PDB entry 1tiw). All eight possible primitive orthorhombic lattices were tested.…”

“…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.…”