Kinetic and Structural Characterization of Tunnel-Perturbing Mutants in Bradyrhizobium japonicum Proline Utilization A

The proline catabolic pathway catalyzes the oxidation of proline to glutamate (Scheme 1). In the first step, proline dehydrogenase (PRODH) uses an FAD cofactor to remove two electrons (as H–) from proline, resulting in Δ1-pyrroline-5-carboxylate (P5C). P5C then undergoes a nonenzymatic hydrolysis, which opens the pyrroline ring to create glutamate-γ-semialdehyde (GSA). Finally, GSA is oxidized to glutamate by the NAD+-dependent P5C dehydrogenase (P5CDH) to complete the overall four-electron oxidation process. Proline and proline metabolism are important for the pathogenicity of Helicobacter pylori and Helicobacter hepaticus,1,2 energy production in procyclic trypanosomes,3,4 and regulation of metabolites linked to pathogenesis in Photorhabdus and Xenorhabdus.5 In humans, inborn errors in proline catabolism lead to hyperprolinemia disorders, and defects in PRODH are linked to schizophrenia.6,7 Also, PRODH is regulated by p53 and has been shown to function in tumor suppression.8 Scheme 1 Overall Reaction Catalyzed by Proline Utilization A (PutA) PRODH and P5CDH are combined into a single polypeptide chain known as proline utilization A (PutA) in Gram-negative bacteria and Corynebacterium.9 The covalent linking of enzymes catalyzing consecutive reactions in a metabolic pathway affords the possibility of substrate channeling; i.e., the intermediate is transferred between the enzymes without equilibrating with the bulk medium. Several physiological benefits of substrate channeling versus free diffusion have been identified. For example, channeling improves kinetic efficiency by decreasing the transit time between active sites and preventing the loss of intermediates.10,11 Thus, channeling enzymes can operate at maximal rates when cellular substrate concentrations are below saturating levels.12 Also, labile intermediates can be concealed from the bulk environment, preventing decay or interaction with other molecules.13,14 Finally, channeling can influence metabolic flux by segregating intermediates from competing pathways.15 Substrate channeling of P5C/GSA in proline catabolism may be necessary to retain proper metabolic flux and avoid metabolic futile cycling.14 In addition, free P5C/GSA is an inhibitor of three different enzymes in Escherichia coli, including glucosamine-6-phosphate synthase, cytidine-5′-triphosphate synthase, and the amidotransferase domain of carbamoyl phosphate synthetase.16−18 P5C has also been shown to form adducts with other metabolites such as oxaloacetic acid, pyruvic acid, and acetoacetic acid.19 Consistent with the physiological importance of controlling the release of P5C/GSA, kinetic studies have firmly established substrate channeling in PutAs. Early studies of Salmonella typhimurium PutA using 14C-labeled proline are consistent with a channeling mechanism.20 More recent steady-state and rapid reaction transient time measurements of PutAs from Bradyrhizobium japonicum (BjPutA) and Geobacter sulfurreducens (GsPutA) also indicate substrate channeling.21,22 Furthermore, a comprehensive analysis of the complete kinetic mechanism of E. coli PutA showed that substrate channeling is rate-limiting, and the rate constant for the channeling step is slowest during the first enzyme turnover and increases with subsequent turnovers, establishing PutA as a new example of a hysteretic enzyme.23 With the kinetic data firmly demonstrating substrate channeling in PutA, the goal of this study is to gain insight into the structural basis of channeling. The crystal structures of BjPutA and GsPutA revealed that the two active sites are separated by a linear distance of 41–45 A, implying that substrate channeling involves substantial movement of the P5C/GSA intermediate.21,22 Analysis of potential channeling pathways predicts a curved, 75 A tunnel that connects the two active sites (Figure ​(Figure11). Figure 1 Tunnel/cavity system of BjPutA. (A) BjPutA protomer with PRODH colored blue, P5CDH pink, and the oligomerization flap green. The FAD and NAD+ are shown as yellow and green sticks, respectively. Catalytic Cys792 of the P5CDH active site is indicated. The ... Here we use site-directed mutagenesis, kinetics, and X-ray crystallography to gain further insight into the structural features that facilitate substrate channeling in BjPutA. Several residues between the two active sites have been mutated in an effort to obstruct molecular traffic. Kinetic and structural analysis of the mutant enzymes shows that channeling is hindered in some of the variants but not others, which provides information about the pathway traversed by the intermediate. Furthermore, steric considerations suggest that GSA is threaded through the tunnel in a linear conformation, with the aldehyde group facing the P5CDH end of the tunnel. This aspect of substrate channeling in PutA might be considered an example of shape selective catalysis.