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Activation studies of the β-carbonic anhydrases from Escherichia coli with amino acids and amines.

ABSTRACT: A β-carbonic anhydrase (CA, EC 4.2.1.1) from the widespread bacterium Escherichia coli (EcoCAβ), encoded by the CynT2 gene, has been investigated for its catalytic properties and enzymatic activation by a panel of amino acids and amines. EcoCAβ showed a significant catalytic activity for the hydration of CO₂ to bicarbonate and a proton, with a kinetic constant k_cat of 5.3 × 10⁵ s^- and a Michaelis-Menten constant K_M of 12.9 mM. The most effective EcoCAβ activators were L- and D-DOPA, L-Tyr, 4-amino-Phe, serotonin and L-adrenaline, with K_As from 2.76 to 10.7 µM. L-His, 2-pyridyl-methylamine, L-Asn and L-Gln were relatively weak activators (K_As from 36.0 to 49.5 µM). D-His, L- and D-Phe, L- and D-Trp, D-Tyr, histamine, dopamine, 2-(aminoethyl)pyridine/piperazine/morpholine, L-Asp, L- and D-Glu have K_As from 11.3 to 23.7 µM. Endogenous CA activators may play a role in bacterial virulence and colonisation of the host.

SUBMITTER: Nocentini A

PROVIDER: S-EPMC7748406 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Activation studies of the β-carbonic anhydrases from Escherichia coli with amino acids and amines.

Nocentini Alessio A Del Prete Sonia S Mastrolorenzo Margaret D MD Donald William A WA Capasso Clemente C Supuran Claudiu T CT

Journal of enzyme inhibition and medicinal chemistry 20201201 1

A β-carbonic anhydrase (CA, EC 4.2.1.1) from the widespread bacterium Escherichia coli (EcoCAβ), encoded by the CynT2 gene, has been investigated for its catalytic properties and enzymatic activation by a panel of amino acids and amines. EcoCAβ showed a significant catalytic activity for the hydration of CO2 to bicarbonate and a proton, with a kinetic constant kcat of 5.3 × 105 s- and a Michaelis-Menten constant KM of 12 ...[more]

PMID: 32576029

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Project description:Non-proteinogenic amino Q7 Q8 acids (npAAs) are valuable building blocks for the development of advanced pharmaceuticals and agrochemicals. The surge in interest in their synthesis is primarily due to the potential to enhance and diversify existing bioactive molecules. This can be achieved by altering these bioactive molecules to improve their effectiveness, reducing resistance compared to their natural counterparts or generating molecules with novel functions. Traditional production of npAAs in native hosts requires specialized conditions and complex cultivation media. Furthermore, these compounds are often found in organisms that challenge genetic manipulation. Thus, the recombinant production of these npAAs in a model organism like Escherichia coli paves the way for groundbreaking advancements in synthetic biology. For the first time, we report the synthesis of npAAs β-methylphenylalanine (BmePhe), β- hydroxyenduracididine (BhEnd), and enduracididine (End) from Streptomyces hygroscopicus in E. coli. Through proteomic and metabolomic analyses, we achieved the production of these compounds from two synthetic operons, comprising five heterologous proteins. Interestingly, we discovered that the exogenous addition of pathway precursors to the E. coli system enhanced the yield of BmePhe by 2.5 times, whereas it concurrently attenuated the production of BhEnd and End, signifying a selective precursor-dependent yield enhancement. The synthetic biology landscape is broadened in this study by expanding the repertoire of amino acids beyond the conventional set of 22 standard proteinogenic amino acids. The biosynthesized npAAs, End, BhEnd, and BmePhe hold promise for engineering proteins with modified functions, integrating into novel metabolites and/or enhancing biological stability and activity. Additionally, these amino acids’ biological production and subsequent purification present an alternative to traditional chemical synthesis methods, paving a direct pathway for pharmacological evaluation.

Dataset Information

Activation studies of the β-carbonic anhydrases from Escherichia coli with amino acids and amines.

Publications

Activation studies of the β-carbonic anhydrases from <i>Escherichia coli</i> with amino acids and amines.

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