Pyruvate‐formate‐lyase‐deactivase and acetyl‐CoA reductase activities of <i>Escherichia coli</i> reside on a polymeric protein particle encoded by <i>adhE</i>

Kessler, Dorothea; Leibrecht, Iris; Knappe, J

doi:10.1016/0014-5793(91)80358-a

Cited by 154 publications

(143 citation statements)

References 12 publications

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“…The results presented herein, however, demonstrate that AdhE and AdhE-His 6 purified from overexpressing cultures do not catalyze the deactivation of active PFL, despite previous reports to the contrary for the wt enzyme [21,22]. Repeated deactivation experiments in the presence of the purported cofactors Fe(II), CoA, and NAD + show that AdhE has no effect on the rate of deactivation of PFL, although both Fe(II) and CoA appear to increase the rate of deactivation.…”

Section: Discussioncontrasting

confidence: 95%

“…For example, we have found the specific activity of AdhE for the ethanol dehydrogenase reaction to be 9.5 U/ mg (for both wild type and histidine-tagged enzyme), as compared to values of 10 U/mg reported previously for wild-type AdhE. [21] All other specific activities were also similar to those reported in the literature, thereby verifying that we have isolated AdhE in its fully functional form.…”

Section: Alcohol Dehydrogenase Activitiessupporting

confidence: 89%

“…The report of PFL deactivation catalyzed by AdhE seemed to resolve the puzzle of the fate of this catalytically essential radical: rather than undergoing oxygenolytic cleavage, as occurs in vitro, the glycyl radical was reported to be catalytically deactivated, or quenched, by AdhE [21,22]. This intriguing deactivation reaction resulted in the conversion of active PFL back to its activatable form, thereby circumventing the irreversible protein cleavage observed in vitro, and presumably allowing the protein to be re-activated upon return to anaerobic conditions.…”

Section: Introductionmentioning

confidence: 99%

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Inactivation of E. coli pyruvate formate-lyase: Role of AdhE and small molecules

Nnyepi

Peng

Broderick

2007

Archives of Biochemistry and Biophysics

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E. coli AdhE has been reported to harbor three distinct enzymatic activities: alcohol dehydrogenase, acetaldehyde-CoA dehydrogenase, and pyruvate formate-lyase (PFL) deactivase. Herein we report on the cloning, expression, and purification of E. coli AdhE, and the re-investigation of its purported enzymatic activities. While both the alcohol dehydrogenase and acetaldehyde-CoA dehydrogenase activities were readily detectible, we were unable to obtain any evidence for catalytic deactivation of PFL by AdhE, regardless of whether the reported cofactors for deactivation (Fe(II), NAD, and CoA) were present. Our results demonstrate that AdhE is not a PFL deactivating enzyme. We have also examined the potential for deactivation of active PFL by small-molecule thiols. Both β-mercaptoethanol and dithiothreitol deactivate PFL efficiently, with the former providing quite rapid deactivation. PFL deactivated by these thiols can be reactivated, suggesting that this deactivation is non-destructive transfer of an H atom equivalent to quench the glycyl radical.

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Section: Discussioncontrasting

confidence: 95%

Section: Alcohol Dehydrogenase Activitiessupporting

confidence: 89%

Section: Introductionmentioning

confidence: 99%

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Inactivation of E. coli pyruvate formate-lyase: Role of AdhE and small molecules

Nnyepi

Peng

Broderick

2007

Archives of Biochemistry and Biophysics

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“…The activity of alcohol dehydrogenase was measured as described by Kessler et al (1991). Cells pellets, harvested as described above, were resuspended in 0.2 mL of 0.1 M MOPS-KOH and permeabilized by vortex mixing with chloroform.…”

Section: Enzyme Activitiesmentioning

confidence: 99%

Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli

Durnin

Clomburg

Yeates

et al. 2009

Biotech & Bioengineering

174

130

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Given its availability, low prices, and high degree of reduction, glycerol has become an ideal feedstock for the production of reduced compounds. The anaerobic fermentation of glycerol by Escherichia coli could be an excellent platform for this purpose but it requires expensive nutrients such as tryptone and yeast extract. In this work, microaerobic conditions were used as a means of eliminating the need for rich nutrients. Availability of low amounts of oxygen enabled redox balance while preserving the ability to synthesize reduced products. A fermentation balance analysis showed $95% recovery of carbon and reducing equivalents. The pathways involved in glycerol dissimilation were identified using different genetic and biochemical approaches. Respiratory (GlpK-GlpD/GlpABC) and fermentative (GldA-DhaKLM) routes mediated the conversion of glycerol to glycolytic intermediates. Although pyruvate formate-lyase (PFL) and pyruvate dehydrogenase contributed to the synthesis of acetyl-CoA from pyruvate, most of the carbon flux proceeded through PFL. The pathways mediating the synthesis of acetate and ethanol were required for the efficient utilization of glycerol. The microaerobic metabolism of glycerol was harnessed by engineering strains for the co-production of ethanol and hydrogen (EH05 [pZSKLMgldA]), and ethanol and formate (EF06 [pZSKLMgldA]). High ethanol yields were achieved by genetic manipulations that reduced the synthesis of byproducts succinate, acetate, and lactate. Co-production of hydrogen required the use of acidic pH while formate co-production was facilitated by inactivation of the enzyme formate-hydrogen lyase. High rates of product synthesis were realized by overexpressing glycerol dehydrogenase (GldA) and dihydroxyacetone kinase (DhaKLM). Engineered strains efficiently produced ethanol and hydrogen and ethanol and formate from glycerol in a minimal medium without rich supplements.

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“…Although this new type of ADH lacked homology with any other previously known ADHs [29], they were demonstrated to utilize ethanol, as well as number of other alcohols, as substrate(s) [6]. In the intervening years, a number of additional microbial enzymes of this iron-activated enzyme type have been described [11,32,50,54,73,83]. Despite the nomenclature of "iron-activated" ADHs, these enzymes are variously activated by a range of divalent cations.…”

Section: Introductionmentioning

confidence: 98%

Differentiation-dependent expression of Adhfe1 in adipogenesis

Kim¹,

Tillison²,

Zhou³

et al. 2007

Archives of Biochemistry and Biophysics

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We have determined that adipocytes are a major site of expression of the transcript for the novel alcohol dehydrogenase (ADH), Adhfe1. Adhfe1 is unique in that the sequence of its encoded protein places it among the iron-activated ADHs. Western blot analysis reveals Adhfe1 encodes a 50 kDa cytoplasmic protein and immunocytochemical staining indicates mitochondrial localization. Adhfe1 transcript exhibits differentiation-dependent expression during in vitro brown and white adipogenesis. Unlike many adipocyte-enriched genes, however, Adhfe1 transcript expression in adipocytes is refractory to TNFα-mediated downregulation. However, use of pharmacological inhibitors reveals PI 3-kinase-mediated signals maintain the basal level of Adhfe1 transcript in 3T3-L1 adipocytes. Tissue profiling studies show Adhfe1 transcript is restricted to white and brown adipose tissues, liver, and kidney. In comparison to C57BL/6 mice, Adhfe1 transcript is downregulated 40% in white adipose tissue of ob/ob obese mice. Further characterization of Adhfe1 should yield new insights into adipocyte function and energy metabolism.

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Pyruvate‐formate‐lyase‐deactivase and acetyl‐CoA reductase activities of Escherichia coli reside on a polymeric protein particle encoded by adhE

Cited by 154 publications

References 12 publications

Inactivation of E. coli pyruvate formate-lyase: Role of AdhE and small molecules

Inactivation of E. coli pyruvate formate-lyase: Role of AdhE and small molecules

Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli

Differentiation-dependent expression of Adhfe1 in adipogenesis

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