The Identification of 5-Hydroxyl-L-norvaline in Cultures of Pyridoxine Auxotrophs of Escherichia coli B

Hill, Robert E.; White, Robert L.; Smith, Kevin C.

doi:10.1021/np50098a007

Cited by 8 publications

(6 citation statements)

References 25 publications

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“…These results indicate that, were HNV to be a part of normal cellular physiology, then ThrRS would likely have evolved the ability to discriminate against it. HNV, however, only appears in bacterial physiology under the unusual condition when pyridoxine auxotrophs of E. coli are starved for pyridoxine . By contrast, ValRS is obliged to discriminate under standard physiological conditions amino acid against isoleucine, which (like HNV) is but one methyl group larger than the cognate.…”

Section: Discussionmentioning

confidence: 99%

“…These results indicate that, were HNV to be a part of normal cellular physiology, then ThrRS would likely have evolved the ability to discriminate against it. HNV, however, only appears in bacterial physiology under the unusual condition when pyridoxine auxotrophs of E. coli are starved for pyridoxine (44). By contrast, ValRS is obliged to discriminate under standard physiological conditions amino acid against isoleucine, which (like HNV) is but one methyl group larger than the cognate.…”

Section: Discussionmentioning

confidence: 99%

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Fidelity Escape by the Unnatural Amino Acid β-Hydroxynorvaline: An Efficient Substrate forEscherichia coliThreonyl-tRNA Synthetase with Toxic Effects on Growth

Minajigi¹,

Deng²,

Francklyn³

2011

Biochemistry

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In all living systems, the fidelity of translation is maintained in part by the editing mechanisms of aminoacyl-tRNA synthetases (ARSs). Some non-proteogenic amino acids, including β-hydroxynorvaline (HNV) are nevertheless efficiently aminoacylated and become incorporated into proteins. To investigate the basis of HNV's ability to function in protein synthesis, the utilization of HNV by E. coli threonyl-tRNA synthetase (ThrRS) was investigated through both in vitro functional experiments and bacterial growth studies. The measured specificity constant (k cat /K M ) for HNV was found to be only 20-30 fold less than that of cognate threonine. The rate of aminoacyl transfer (10.4 s −1 ) was 10-fold higher than the multiple turnover k cat value (1 s −1 ), indicating that, as for cognate threonine, amino acid activation is likely to be the rate-limiting step. Like non-cognate serine, HNV enhances the ATPase function of the synthetic site, at a rate not increased by non-aminoacylatable (3′-dA76) tRNA. ThrRS also failed to exhibit post-transfer editing activity against HNV. In growing bacteria, the addition of HNV dramatically suppressed growth rates, which indicates either negative phenotypic consequences associated with its incorporation into protein, or inhibition of an unidentified metabolic reaction. The inability of wild ThrRS to prevent utilization of HNV as a substrate illustrates that, for at least one ARS, the naturally occurring enzyme lacks the capability to effectively discriminate against non-proteogenic amino acids that are not encountered under normal physiological conditions. Other examples of 'fidelity escape' in the ARSs may serve as useful starting points in the design of ARSs with specificity for unnatural amino acids.Aminoacyl-tRNA synthetases establish the fidelity of the genetic code in a two-step aminoacylation reaction, covalently linking specific amino acids to their corresponding tRNAs. In the first step, amino acid is condensed with ATP to form the aminoacyl adenylate (adenylation), while in the second step, the amino acid moiety of adenylate is transferred to the A76 of tRNA (aminoacyl transfer), along with the release of pyrophosphate (PPi) and AMP, respectively.

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Section: Discussionmentioning

confidence: 99%

“…These results indicate that, were HNV to be a part of normal cellular physiology, then ThrRS would likely have evolved the ability to discriminate against it. HNV, however, only appears in bacterial physiology under the unusual condition when pyridoxine auxotrophs of E. coli are starved for pyridoxine (44). By contrast, ValRS is obliged to discriminate under standard physiological conditions amino acid against isoleucine, which (like HNV) is but one methyl group larger than the cognate.…”

Section: Discussionmentioning

confidence: 99%

Fidelity Escape by the Unnatural Amino Acid β-Hydroxynorvaline: An Efficient Substrate forEscherichia coliThreonyl-tRNA Synthetase with Toxic Effects on Growth

Minajigi¹,

Deng²,

Francklyn³

2011

Biochemistry

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“…Moreover, the CBD-induced changes in the action potential of fast-spiking CA1 pyramidal neurons closely resemble those produced by paxilline. 13,14 Consequently, we can postulate that the blockade of BK channels represents an additional mechanism through which CBD exerts its therapeutic antiepileptic effects. The low concentration range at which CBD inhibits BK channels suggests a potential synergistic effect with other previously described anticonvulsant mechanisms, ultimately reducing neuronal excitability.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…In this regard, it has been demonstrated that CBD diminishes the firing frequency in rat CA1 hippocampal neurons in brain slices, in part by prolonging the duration of action potentials. 13 This effect could be explained by the inhibition of fast voltageoperated K + channels involved in action potential repolarization. Therefore, we hypothesize that CBD's anticonvulsant activity might be linked to the inhibition of the large conductance voltage-and Ca 2+ -operated K + channel, known as the BK channel, which is involved in firing behavior of CA1 hippocampal pyramidal cells.…”

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confidence: 99%

Direct Inhibition of BK Channels by Cannabidiol, One of the Principal Therapeutic Cannabinoids Derived from Cannabis sativa

Monat,

Altieri,

Enrique

et al. 2024

J. Nat. Prod.

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Cannabidiol (CBD), one of the main Cannabis sativa bioactive compounds, is utilized in the treatment of major epileptic syndromes. Its efficacy can be attributed to a multimodal mechanism of action that includes, as potential targets, several types of ion channels. In the brain, CBD reduces the firing frequency in rat hippocampal neurons, partly prolonging the duration of action potentials, suggesting a potential blockade of voltage-operated K + channels. We postulate that this effect might involve the inhibition of the large-conductance voltage-and Ca 2+operated K + channel (BK channel), which plays a role in the neuronal action potential's repolarization. Thus, we assessed the impact of CBD on the BK channel activity, heterologously expressed in HEK293 cells. Our findings, using the patch-clamp technique, revealed that CBD inhibits BK channel currents in a concentration-dependent manner with an IC 50 of 280 nM. The inhibition is through a direct interaction, reducing both the unitary conductance and voltage-dependent activation of the channel. Additionally, the cannabinoid significantly delays channel activation kinetics, indicating stabilization of the closed state. These effects could explain the changes induced by CBD in action potential shape and duration, and they may contribute to the observed anticonvulsant activity of this cannabinoid.

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“…A potential candidate substrate in C. reinhardtii for bacterial ODCs to yield 4‐aminobutanol by decarboxylation is L‐pentahomoserine (5‐hydroxy‐L‐norvaline, CAS 6152‐89‐2). It was found in bacteria (Hill et al ., 1993) and plants (Carmo‐Silva et al ., 2009) before, but its existence in C. reinhardtii is unclear. Since it represents the reduced form of the proline biosynthesis intermediate glutamate‐5‐semialdehyde (Figure 1a), some species (e.g.…”

Section: Resultsmentioning

confidence: 99%

Advanced pathway engineering for phototrophic putrescine production

Freudenberg

Wittemeier

Einhaus

et al. 2022

Plant Biotechnology Journal

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The polyamine putrescine (1,4-diaminobutane) contributes to cellular fitness in most organisms, where it is derived from the amino acids ornithine or arginine. In the chemical industry, putrescine serves as a versatile building block for polyamide synthesis. The green microalga Chlamydomonas reinhardtii accumulates relatively high putrescine amounts, which, together with recent advances in genetic engineering, enables the generation of a powerful green cell factory to promote sustainable biotechnology for base chemical production. Here, we report a systematic investigation of the native putrescine metabolism in C. reinhardtii, leading to the first CO 2 -based bio-production of putrescine, by employing modern synthetic biology and metabolic engineering strategies. A CRISPR/Cas9-based knockout of key enzymes of the polyamine biosynthesis pathway identified ornithine decarboxylase 1 (ODC1) as a gatekeeper for putrescine accumulation and demonstrated that the arginine decarboxylase (ADC) route is likely inactive and that amine oxidase 2 (AMX2) is mainly responsible for putrescine degradation in C. reinhardtii. A 4.5-fold increase in cellular putrescine levels was achieved by engineered overexpression of potent candidate ornithine decarboxylases (ODCs). We identified unexpected substrate promiscuity in two bacterial ODCs, which exhibited co-production of cadaverine and 4aminobutanol. Final pathway engineering included overexpression of recombinant arginases for improved substrate availability as well as functional knockout of putrescine degradation, which resulted in a 10-fold increase in cellular putrescine titres and yielded 200 mg/L in phototrophic high cell density cultivations after 10 days.

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The Identification of 5-Hydroxyl-L-norvaline in Cultures of Pyridoxine Auxotrophs of Escherichia coli B

Cited by 8 publications

References 25 publications

Fidelity Escape by the Unnatural Amino Acid β-Hydroxynorvaline: An Efficient Substrate forEscherichia coliThreonyl-tRNA Synthetase with Toxic Effects on Growth

Fidelity Escape by the Unnatural Amino Acid β-Hydroxynorvaline: An Efficient Substrate forEscherichia coliThreonyl-tRNA Synthetase with Toxic Effects on Growth

Direct Inhibition of BK Channels by Cannabidiol, One of the Principal Therapeutic Cannabinoids Derived from Cannabis sativa

Advanced pathway engineering for phototrophic putrescine production

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