Ranjan Singh

Publications

• The Three-Dimensional Structural Basis of Type II Hyperprolinemia.

  Srivastava D, Singh RK, Moxley MA, Henzl MT, Becker DF, Tanner JJ

  journal of molecular biology. 04/2012;

  Type II hyperprolinemia is an autosomal recessive disorder caused by a deficiency in Δ1- pyrroline-5-carboxylate dehydrogenase (P5CDH, aka ALDH4A1), the aldehyde dehydrogenase that catalyzes the oxidation of glutamate semialdehyde to glutamate. Here we report the first structure of human P5CDH and i... [more] Type II hyperprolinemia is an autosomal recessive disorder caused by a deficiency in Δ1- pyrroline-5-carboxylate dehydrogenase (P5CDH, aka ALDH4A1), the aldehyde dehydrogenase that catalyzes the oxidation of glutamate semialdehyde to glutamate. Here we report the first structure of human P5CDH and investigate the impact of the hyperprolinemia-associated mutation of Ser352 to Leu on the structure and catalytic properties of the enzyme. The 2.5 Å resolution crystal structure of human P5CDH was determined using experimental phasing. Structures of the mutant enzymes S352A (2.4 Å) and S352L (2.85 Å) were determined to elucidate the structural consequences of altering Ser352. Structures of the 93%-identical mouse P5CDH complexed with sulfate ion (1.3 Å resolution), glutamate (1.5 Å), and NAD(+) (1.5 Å) were determined to obtain high resolution views of the active site. Together, the structures show that Ser352 occupies a hydrophilic pocket and is connected via water-mediated hydrogen bonds to catalytic Cys348. Mutation of Ser352 to Leu is shown to abolish catalytic activity and eliminate NAD+binding. Analysis of the S352A mutant shows that these functional defects are caused by the introduction of the nonpolar Leu352 side chain rather than the removal of the Ser352 hydroxyl. The S352L structure shows that the mutation induces a dramatic 8-Å rearrangement of the catalytic loop. Because of this conformational change, Ser349 is not positioned to interact with the aldehyde substrate, conserved Glu447 is no longer poised to bind NAD+, and Cys348 faces the wrong direction for nucleophilic attack. These structural alterations render the enzyme inactive.
• 3.74

  Impact points

  Unique structural features and sequence motifs of proline utilization A (PutA).

  Ranjan K Singh, John J Tanner

  Frontiers in bioscience : a journal and virtual library. 01/2012; 17:556-68.

  Proline utilization A proteins (PutAs) are bifunctional enzymes that catalyze the oxidation of proline to glutamate using spatially separated proline dehydrogenase and pyrroline-5-carboxylate dehydrogenase active sites. Here we use the crystal structure of the minimalist PutA from Bradyrhizobium jap... [more] Proline utilization A proteins (PutAs) are bifunctional enzymes that catalyze the oxidation of proline to glutamate using spatially separated proline dehydrogenase and pyrroline-5-carboxylate dehydrogenase active sites. Here we use the crystal structure of the minimalist PutA from Bradyrhizobium japonicum (BjPutA) along with sequence analysis to identify unique structural features of PutAs. This analysis shows that PutAs have secondary structural elements and domains not found in the related monofunctional enzymes. Some of these extra features are predicted to be important for substrate channeling in BjPutA. Multiple sequence alignment analysis shows that some PutAs have a 17-residue conserved motif in the C-terminal 20-30 residues of the polypeptide chain. The BjPutA structure shows that this motif helps seal the internal substrate-channeling cavity from the bulk medium. Finally, it is shown that some PutAs have a 100-200 residue domain of unknown function in the C-terminus that is not found in minimalist PutAs. Remote homology detection suggests that this domain is homologous to the oligomerization beta-hairpin and Rossmann fold domain of BjPutA.
• 5.33

  Impact points

  Small-angle X-ray scattering studies of the oligomeric state and quaternary structure of the trifunctional proline utilization A (PutA) flavoprotein from Escherichia coli.

  Ranjan K Singh, John D Larson, Weidong Zhu, Robert P Rambo, Greg L Hura, Donald F Becker, John J Tanner

  The Journal of biological chemistry. 12/2011; 286(50):43144-53.

  The trifunctional flavoprotein proline utilization A (PutA) links metabolism and gene regulation in Gram-negative bacteria by catalyzing the two-step oxidation of proline to glutamate and repressing transcription of the proline utilization regulon. Small-angle x-ray scattering (SAXS) and domain dele... [more] The trifunctional flavoprotein proline utilization A (PutA) links metabolism and gene regulation in Gram-negative bacteria by catalyzing the two-step oxidation of proline to glutamate and repressing transcription of the proline utilization regulon. Small-angle x-ray scattering (SAXS) and domain deletion analysis were used to obtain solution structural information for the 1320-residue PutA from Escherichia coli. Shape reconstructions show that PutA is a symmetric V-shaped dimer having dimensions of 205 × 85 × 55 Å. The particle consists of two large lobes connected by a 30-Å diameter cylinder. Domain deletion analysis shows that the N-terminal DNA-binding domain mediates dimerization. Rigid body modeling was performed using the crystal structure of the DNA-binding domain and a hybrid x-ray/homology model of residues 87-1113. The calculations suggest that the DNA-binding domain is located in the connecting cylinder, whereas residues 87-1113, which contain the two catalytic active sites, reside in the large lobes. The SAXS data and amino acid sequence analysis suggest that the Δ(1)-pyrroline-5-carboxylate dehydrogenase domains lack the conventional oligomerization flap, which is unprecedented for the aldehyde dehydrogenase superfamily. The data also provide insight into the function of the 200-residue C-terminal domain. It is proposed that this domain serves as a lid that covers the internal substrate channeling cavity, thus preventing escape of the catalytic intermediate into the bulk medium. Finally, the SAXS model is consistent with a cloaking mechanism of gene regulation whereby interaction of PutA with the membrane hides the DNA-binding surface from the put regulon thereby activating transcription.