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Kinetic and structural characterization of tunnel-perturbing mutants in Bradyrhizobium japonicum proline utilization A.

ABSTRACT: Proline utilization A from Bradyrhizobium japonicum (BjPutA) is a bifunctional flavoenzyme that catalyzes the oxidation of proline to glutamate using fused proline dehydrogenase (PRODH) and ?(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Recent crystal structures and kinetic data suggest an intramolecular channel connects the two active sites, promoting substrate channeling of the intermediate ?(1)-pyrroline-5-carboxylate/glutamate-?-semialdehyde (P5C/GSA). In this work, the structure of the channel was explored by inserting large side chain residues at four positions along the channel in BjPutA. Kinetic analysis of the different mutants revealed replacement of D779 with Tyr (D779Y) or Trp (D779W) significantly decreased the overall rate of the PRODH-P5CDH channeling reaction. X-ray crystal structures of D779Y and D779W revealed that the large side chains caused a constriction in the central section of the tunnel, thus likely impeding the travel of P5C/GSA in the channel. The D779Y and D779W mutants have PRODH activity similar to that of wild-type BjPutA but exhibit significantly lower P5CDH activity, suggesting that exogenous P5C/GSA enters the channel upstream of Asp779. Replacement of nearby Asp778 with Tyr (D778Y) did not impact BjPutA channeling activity. Consistent with the kinetic results, the X-ray crystal structure of D778Y shows that the main channel pathway is not impacted; however, an off-cavity pathway is closed off from the channel. These findings provide evidence that the off-cavity pathway is not essential for substrate channeling in BjPutA.

SUBMITTER: Arentson BW

PROVIDER: S-EPMC4131897 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Kinetic and structural characterization of tunnel-perturbing mutants in Bradyrhizobium japonicum proline utilization A.

Arentson Benjamin W BW Luo Min M Pemberton Travis A TA Tanner John J JJ Becker Donald F DF

Biochemistry 20140730 31

Proline utilization A from Bradyrhizobium japonicum (BjPutA) is a bifunctional flavoenzyme that catalyzes the oxidation of proline to glutamate using fused proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Recent crystal structures and kinetic data suggest an intramolecular channel connects the two active sites, promoting substrate channeling of the intermediate Δ(1)-pyrroline-5-carboxylate/glutamate-γ-semialdehyde (P5C/GSA). In this work, the structur ...[more]

PMID: 25046425

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Project description:PutA is a bifunctional flavoenzyme in bacteria that catalyzes the four-electron oxidation of proline to glutamate. In certain prokaryotes such as Escherichia coli, PutA is also a transcriptional repressor of the proline utilization (put) genes and thus is trifunctional. In this work, we have begun to assess differences between bifunctional and trifunctional PutA enzymes by examining the PutA protein from Bradyrhizobium japonicum (BjPutA). Primary structure analysis of BjPutA shows it lacks the DNA-binding domain of E. coli PutA (EcPutA). Consistent with this prediction, purified BjPutA does not exhibit DNA-binding activity in native gel mobility shift assays with promoter regions of the putA gene from B. japonicum. The catalytic and redox properties of BjPutA were characterized and a reduction potential (E(m)) value of -0.132 V (pH 7.5) was determined for the bound FAD/FADH(2) couple in BjPutA that is significantly more negative ( approximately 55 mV) than the E(m) for EcPutA-bound FAD. The more negative E(m) value thermodynamically limits proline reduction of the FAD cofactor in BjPutA. In the presence of phospholipids, reduction of BjPutA is stimulated, suggesting lipids influence the FAD redox environment. Accordingly, an E(m) value of -0.114 V (pH 7.5) was determined for BjPutA-bound FAD in the presence of polar lipids. The molecular basis for the lower reduction potential of FAD in BjPutA relative to EcPutA was explored by site-directed mutagenesis. Amino acid sequence alignment between BjPutA and EcPutA indicates only one difference in active site residues near the isoalloxazine ring of FAD: Val402 in EcPutA is substituted at the analogous position in BjPutA with Ala310. Replacement of A310 by Val in the BjPutA mutant A310V raised the reduction potential of bound FAD relative to wild-type BjPutA to an E(m) value of -0.09 V (pH 7.5). The >40-mV positive shift in the potential of the BjPutA mutant A310V suggests that the corresponding Val residue in EcPutA helps poise the FAD redox potential for thermodynamically favored proline reduction thereby allowing EcPutA to be efficiently regulated by proline availability. Limited proteolysis of BjPutA under reducing conditions shows FAD reduction does not influence BjPutA conformation indicating further that the redox dependent regulation observed with EcPutA may be limited to trifunctional PutA homologues.

Dataset Information

Kinetic and structural characterization of tunnel-perturbing mutants in Bradyrhizobium japonicum proline utilization A.

Publications

Kinetic and structural characterization of tunnel-perturbing mutants in Bradyrhizobium japonicum proline utilization A.

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