Effective production of Pro–Gly by mutagenesis of l-amino acid ligase

Kino, Haruka; Nakajima, Shota; Arai, Tomomi; Kino, Kuniki

doi:10.1016/j.jbiosc.2016.01.014

Cited by 13 publications

(9 citation statements)

References 12 publications

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“…If this is found to apply to other ATP-grasp enzymes, we predict that using K + concentrations that more closely reflect physiological conditions will allow more accurate characterization of inhibitors in vitro. More recently, L-amino acid ligases (LALs) have also been investigated for the industrial production of a range of products, including antibiotics and peptides for use in healthcare and consumable products (28)(29)(30)(31). Despite many ATP-grasp enzymes sharing high similarity to Ddl, to our knowledge the activation of LALs by MVCs has not been investigated.…”

Section: Discussionmentioning

confidence: 99%

d-Alanine–d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations

Pederick

Thompson

Bell

et al. 2020

Journal of Biological Chemistry

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The ATP-grasp superfamily of enzymes shares an atypical nucleotide-binding site known as the ATP-grasp fold. These enzymes are involved in many biological pathways in all domains of life. One ATP-grasp enzyme, d-alanine–d-alanine ligase (Ddl), catalyzes ATP-dependent formation of the d-alanyl–d-alanine dipeptide essential for bacterial cell wall biosynthesis and is therefore an important antibiotic drug target. Ddl is activated by the monovalent cation (MVC) K+, but despite its clinical relevance and decades of research, how this activation occurs has not been elucidated. We demonstrate here that activating MVCs bind adjacent to the active site of Ddl from Thermus thermophilus and used a combined biochemical and structural approach to characterize MVC activation. We found that TtDdl is a type II MVC-activated enzyme, retaining activity in the absence of MVCs. However, the efficiency of TtDdl increased ∼20-fold in the presence of activating MVCs, and it was maximally activated by K+ and Rb+ ions. A strict dependence on ionic radius of the MVC was observed, with Li+ and Na+ providing little to no TtDdl activation. To understand the mechanism of MVC activation, we solved crystal structures of TtDdl representing distinct catalytic stages in complex with K+, Rb+, or Cs+. Comparison of these structures with apo TtDdl revealed no evident conformational change on MVC binding. Of note, the identified MVC binding site is structurally conserved within the ATP-grasp superfamily. We propose that MVCs activate Ddl by altering the charge distribution of its active site. These findings provide insight into the catalytic mechanism of ATP-grasp enzymes.

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Section: Discussionmentioning

confidence: 99%

d-Alanine–d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations

Pederick

Thompson

Bell

et al. 2020

Journal of Biological Chemistry

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“…A ceiling to the final product concentration could also be imposed by the RizA enzyme itself and thus be addressed by protein engineering. This was done for the LAL TabS in which the double mutant S85T/H294D reached a 1.6-fold higher Pro-Gly concentration after 20 h than the wild type [27].…”

Section: Time Course Of Formation Of Arg-ser Arg-ala Arg-gly and Arg-argmentioning

confidence: 99%

Recombinant Production of Arginyl Dipeptides by l-Amino Acid Ligase RizA Coupled with ATP Regeneration

et al. 2021

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Arginyl dipeptides like Arg-Ser, Arg-Ala, and Arg-Gly are salt-taste enhancers and can potentially be used to reduce the salt content of food. The l-amino acid ligase RizA from B. subtilis selectively synthesizes arginyl dipeptides. However, industrial application is prevented by the high cost of the cofactor adenosine triphosphate (ATP). Thus, a coupled reaction system was created consisting of RizA and acetate kinase (AckA) from E. coli providing ATP regeneration from acetyl phosphate. Both enzymes were recombinantly produced in E. coli and purified by affinity chromatography. Biocatalytic reactions were varied and analyzed by RP-HPLC with fluorescence detection. Under optimal conditions the system produced up to 5.9 g/L Arg-Ser corresponding to an ATP efficiency of 23 g Arg-Ser per gram ATP. Using similar conditions with alanine or glycine as second amino acid, 2.6 g/L Arg-Ala or 2.4 g/L Arg Gly were produced. The RizA/AckA system selectively produced substantial amounts of arginyl dipeptides while minimizing the usage of the expensive ATP.

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“…A number of functional peptides was synthesised, including l ‐arginyl‐ l ‐phenylalanine and l ‐leucyl‐ l ‐isoleucine in 62% and 77% yield and which are reported to have antihypertensive and antidepressive properties, respectively. Further structure‐guided mutation of TabS resulted in the double mutant S85T/H294D, which displayed improved activity with respect to the synthesis of the salt taste enhancing dipeptide Pro‐Gly …”

Section: Atp‐dependent Systemsmentioning

confidence: 99%

Enzyme‐Catalysed Synthesis of Secondary and Tertiary Amides

Petchey

Grogan

2019

Adv Synth Catal

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Thes ynthesis of the amide bondb etween an amine andacarboxylic acid is one of the most significant reactions in industrial pharmaceutical synthesis.D espite the apparents implicityo fs ynthetic methodsf or amide bond formation, many of these are disadvantaged by their requirementf or toxic or hazardous reagents for the activation of the acid component, or poora tom economy resulting from the need for stoichiometric amountso fc oupling reagents.I nt his context, biocatalysis hase merged as an alternative catalytic method for amide bond formation, presenting the advantages of both environmentally benign reagents andc onditions and also atom economy.I nthis review we detail developments in the enzyme-catalysed preparation of secondary andt ertiary amides for the synthesis of pharmaceutical-type molecules andr eview the applications of hydrolases,s uch as lipasesa nd penicillina cylases, to these reactions.W ea lso summarise the activity of ATP-dependent enzymesf or amide bondf ormation and assess their potentialf or the preparative synthesis of amidesf rom carboxylic acids and amines in aqueous media.

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Effective production of Pro–Gly by mutagenesis of l-amino acid ligase

Cited by 13 publications

References 12 publications

d-Alanine–d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations

d-Alanine–d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations

Recombinant Production of Arginyl Dipeptides by l-Amino Acid Ligase RizA Coupled with ATP Regeneration

Enzyme‐Catalysed Synthesis of Secondary and Tertiary Amides

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