Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenase and Δ1-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline Utilization A (PutA)

Moxley, Michael A.; Sanyal, Nikhilesh; Krishnan, Navasona; Tanner, John J.; Becker, Donald F.

doi:10.1074/jbc.m113.523704

Cited by 30 publications

(72 citation statements)

References 52 publications

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“…The observed lag time is greater than that observed for PutA. The lag times reported for PutA enzymes thus far range from no apparent lag to 23 s for E. coli PutA (8,9,11). The longer lag time of the TtPRODH- (50).…”

Section: Discussionmentioning

confidence: 66%

“…Blockage of the tunnel in B. japonicum PutA by mutagenesis abrogates channeling, which indicates that the tunnel functions as the conduit for the intermediate P5C/GSA (10). Furthermore, a substrate channeling mechanism is supported by kinetic studies of PutA from Escherichia coli (11) and Salmonella typhimurium (12) indicating that a general feature of PutA enzymes is direct transfer of P5C between the PRODH and P5CDH active sites.…”

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confidence: 63%

“…Very recently, P5C was proposed to act as a signaling metabolite linking proline metabolism with lipid oxidation in Caenorhabditis elegans (22). A more compelling reason for channeling P5C/GSA, however, may be to avoid futile cycling (11,12,23,24). Cycling between proline catabolism and proline biosynthesis would be energetically costly for the cell.…”

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confidence: 99%

“…) using a 0.15 cm path length on a Hi-Tech Scientific SF-61DX2 stopped-flow instrument (TgK Scientific) at 25°C (11). The kinetic parameters K m and k cat were estimated by fitting initial velocities to the MichaelisMenten equation (35,36).…”

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confidence: 99%

“…Assays were performed using 0.5 M P5CDH, 0.2 mM NAD ϩ , 50 mM potas- sium phosphate (pH 7.5), 25 mM NaCl and varying L-P5C (1-300 M). The concentration of L-P5C is considered to be half the total (DL)-P5C concentration (11,12). The kinetic parameters K m and k cat were estimated by fitting initial velocities to the Michaelis-Menten equation (35,36 …”

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confidence: 99%

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First Evidence for Substrate Channeling between Proline Catabolic Enzymes

Sanyal

Arentson

Luo

et al. 2015

Journal of Biological Chemistry

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Background: PRODH and P5CDH from Thermus thermophilus are monofunctional enzymes in proline catabolism. Results: Steady-state kinetics and intermediate trapping data show the PRODH and P5CDH reactions are coupled by a channeling step. Conclusion: Substrate channeling in monofunctional enzymes is achieved via weak interactions. Significance: Evidence for substrate channeling between monofunctional proline catabolic enzymes is shown and confirms the Rosetta Stone hypothesis.

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Section: Discussionmentioning

confidence: 66%

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confidence: 63%

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confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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First Evidence for Substrate Channeling between Proline Catabolic Enzymes

Sanyal

Arentson

Luo

et al. 2015

Journal of Biological Chemistry

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Engineering substrate channeling in biosystems for improved efficiency

Wang

Xin

Liang

et al. 2018

J of Chemical Tech & Biotech

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The efficiency of a biochemical reaction is contextually regulated in the landscape of the metabolic network of a cell. The availability and activity of the corresponding enzyme are maintained at a level that favors the overall fitness of the cell, constituting a big issue for biotechnology application that a substantial accumulation of specific product is principally expected. Substrate channeling mechanism naturally existing in multiple active site enzymes, enhances the catalytic efficiency of the involved enzymes via the proximity effect enabled by the'evolutionally optimized'orientation of the enzymes. In metabolic engineering or in vitro cascade biocatalysis, improved efficiency can be obtained when the involved enzymes are rationally organized, to simulate substrate channeling mechanism in artificially constructed systems. In this paper, we reviewed the available approaches adopted in constructing in vitro cascade biocatalysis or in vivo engineered pathways for the production of valuable products. Among these approaches, assembling enzymes onto the designed scaffolds (protein, DNA or RNA) has been proved effective in many metabolic engineering studies. Encapsulating enzymes in virus capsid or encapsulin can be used to create specialized 'organelle-like' microreactors in cell by simulating the naturally existing bacterial microcompartment. Assembling enzymes on lipid droplet or thylakoid membrane of chloroplasts, will be of particular significance in practice in plant cell bioreactors. Inclusion of a feedback mechanism as an addition to these engineered systems may promise further increased efficiency.

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The urea carboxylase and allophanate hydrolase activities of urea amidolyase are functionally independent

2016

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Urea amidolyase (UAL) is a multifunctional biotin-dependent enzyme that contributes to both bacterial and fungal pathogenicity by catalyzing the ATP-dependent cleavage of urea into ammonia and CO 2 . UAL is comprised of two enzymatic components: urea carboxylase (UC) and allophanate hydrolase (AH). These enzyme activities are encoded on separate but proximally related genes in prokaryotes while, in most fungi, they are encoded by a single gene that produces a fusion enzyme on a single polypeptide chain. It is unclear whether the UC and AH activities are connected through substrate channeling or other forms of direct communication. Here, we use multiple biochemical approaches to demonstrate that there is no substrate channeling or interdomain/intersubunit communication between UC and AH. Neither stable nor transient interactions can be detected between prokaryotic UC and AH and the catalytic efficiencies of UC and AH are independent of one another. Furthermore, an artificial fusion of UC and AH does not significantly alter the AH enzyme activity or catalytic efficiency. These results support the surprising functional independence of AH from UC in both the prokaryotic and fungal UAL enzymes and serve as an important reminder that the evolution of multifunctional enzymes through gene fusion events does not always correlate with enhanced catalytic function.

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Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenase and Δ1-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline Utilization A (PutA)

Cited by 30 publications

References 52 publications

First Evidence for Substrate Channeling between Proline Catabolic Enzymes

First Evidence for Substrate Channeling between Proline Catabolic Enzymes

Engineering substrate channeling in biosystems for improved efficiency

The urea carboxylase and allophanate hydrolase activities of urea amidolyase are functionally independent

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