Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides

Wood, Alexander James; Weise, Nicholas J.; Frampton, Joseph D.; Dunstan, Mark S.; Hollas, Michael A.; Derrington, Sasha R.; Lloyd, Richard C.; Quaglia, Daniela; Parmeggiani, Fabio; Leys, D.; Turner, Nicholas J.; Flitsch, Sabine L.

doi:10.1002/anie.201707918

Cited by 77 publications

(67 citation statements)

References 30 publications

(73 reference statements)

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“…As already reported by others, the replacement of the phosphopantetheine binding site Ser 689 in Ni CAR by Ala resulted in an inactive variant (Venkitasubramanian et al, 2007 ; Wood et al, 2017 ). The same is true for the equivalent exchange of Ser 595 in Nc CAR [Table 2 , entry 7; or core motif T (core 6) in Marahiel et al ( 1997 )], confirming that this residue serves as the phosphopantetheine attachment site and is indispensable for CAR activity.…”

Section: Discussionsupporting

confidence: 55%

Identification of Key Residues for Enzymatic Carboxylate Reduction

et al. 2018

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Carboxylate reductases (CARs, E.C. 1.2.1.30) generate aldehydes from their corresponding carboxylic acid with high selectivity. Little is known about the structure of CARs and their catalytically important amino acid residues. The identification of key residues for carboxylate reduction provides a starting point to gain deeper understanding of enzymatic carboxylate reduction. A multiple sequence alignment of CARs with confirmed activity recently identified in our lab and from the literature revealed a fingerprint of conserved amino acids. We studied the function of conserved residues by multiple sequence alignments and mutational replacements of these residues. In this study, single-site alanine variants of Neurospora crassa CAR were investigated to determine the contribution of conserved residues to the function, expressability or stability of the enzyme. The effect of amino acid replacements was investigated by analyzing enzymatic activity of the variants in vivo and in vitro. Supported by molecular modeling, we interpreted that five of these residues are essential for catalytic activity, or substrate and co-substrate binding. We identified amino acid residues having significant impact on CAR activity. Replacement of His 237, Glu 433, Ser 595, Tyr 844, and Lys 848 by Ala abolish CAR activity, indicating their key role in acid reduction. These results may assist in the functional annotation of CAR coding genes in genomic databases. While some other conserved residues decreased activity or had no significant impact, four residues increased the specific activity of NcCAR variants when replaced by alanine. Finally, we showed that NcCAR wild-type and mutants efficiently reduce aliphatic acids.

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Section: Discussionsupporting

confidence: 55%

Identification of Key Residues for Enzymatic Carboxylate Reduction

et al. 2018

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“…16 a was also isolated, although only in 15 %y ield owing to complications in extraction of this indole product. These results compare favorably with the application of CAR-A-PCP to preparative amide bond formation, [9] since 100 equivalents of amine donor were required for those reactions, compared to the low ratios reported here.The difference may reflect of the active participation of McbA in amine binding and catalysis of amidation, which was not yet established for the CAR-A-PCP reaction. Ther esults offer promise for the application of McbA-type enzymes in preparative biocatalyzed reactions,p articularly if ATPr ecycling methods, currently as ubject of significant interest in biocatalysis, [25] were to be employed.…”

supporting

confidence: 67%

“…Hydrolases, such as N‐acylases and lipases, have been employed for amide bond formation and are attractive in terms of their simplicity and efficiency, but they often require ester substrates and suffer from poor substrate scope. More recently, Flitsch and co‐workers have recruited the adenylate‐forming ability of a carboxylate reductase (CAR) adenylation‐domain‐plus‐peptidyl carrier protein (CAR‐A‐PCP) to the formation of amides, although a 100‐fold excess of amine was required to drive reactions to high conversions …”

Section: Methodsmentioning

confidence: 99%

“…[23,24] Thea midation conformer of McbA superimposes well with the thiolation conformer of 4-CBL (PDB ID:3CW9, 2.0 over 430 Ca atoms) and also the "A"d omain in the structure of CAR-A-PCP (PDB ID:5MSS,2 .5 over 402), the construct used in amide bond forming reactions described previously. [9] Thestructure of 4-CBL in the thiolation state,in complex with 4-chlorophenacyl-CoA (4-CPA-CoA, PDB ID:3CW9;F igure S3a) [23] identifies the binding site for the thiolation product, and permits acomparison with the equivalent region of McbA ( Figure S3b) Thes urfaces of the enzymes show that positively charged residues in 4-CBL, such as R87, which interact with the phosphates in 4-CPA-CoA, are absent in McbA;i ndeed the entrance to the active site features three residues with carboxylate side chains (D463, E221 and E400), which may favor interaction with incoming amine substrates.…”

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confidence: 99%

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The Broad Aryl Acid Specificity of the Amide Bond Synthetase McbA Suggests Potential for the Biocatalytic Synthesis of Amides

et al. 2018

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Amide bond formation is one of the most important reactions in pharmaceutical synthetic chemistry. The development of sustainable methods for amide bond formation, including those that are catalyzed by enzymes, is therefore of significant interest. The ATP‐dependent amide bond synthetase (ABS) enzyme McbA, from Marinactinospora thermotolerans, catalyzes the formation of amides as part of the biosynthetic pathway towards the marinacarboline secondary metabolites. The reaction proceeds via an adenylate intermediate, with both adenylation and amidation steps catalyzed within one active site. In this study, McbA was applied to the synthesis of pharmaceutical‐type amides from a range of aryl carboxylic acids with partner amines provided at 1–5 molar equivalents. The structure of McbA revealed the structural determinants of aryl acid substrate tolerance and differences in conformation associated with the two half reactions catalyzed. The catalytic performance of McbA, coupled with the structure, suggest that this and other ABS enzymes may be engineered for applications in the sustainable synthesis of pharmaceutically relevant (chiral) amides.

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“…For example, the conversion of dodecane to 800 mg/liter 1-dodecanol and 79 mg/liter 1,12-dodecanediol using AlkB monooxygenase or CYP153A with heterologous redox partners was reported (14)(15)(16)32). In addition, only the small-scale biotransformation of alkanamine was tested (12,33). In this study, we improved the levels of production to 1.5 g/liter 1-dodecanol and 3.76 g/liter 1,12 dodecanediol.…”

Section: Discussionmentioning

confidence: 99%

Production of 1-Dodecanol, 1-Tetradecanol, and 1,12-Dodecanediol through Whole-Cell Biotransformation in Escherichia coli

Hsieh

Wang

Lai

et al. 2018

Appl Environ Microbiol

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Medium- and long-chain 1-alkanol and α,ω-alkanediols are used in personal care products, in industrial lubricants, and as precursors for polymers synthesized for medical applications. The industrial production of α,ω-alkanediols by alkane hydroxylation primarily occurs at high temperature and pressure using heavy metal catalysts. However, bioproduction has recently emerged as a more economical and environmentally friendly alternative. Among alkane monooxygenases, CYP153A from VT8 (CYP153A ; the strain is also known as VT8) possesses low overoxidation activity and high regioselectivity and thus has great potential for use in terminal hydroxylation. However, the application of CYP153A is limited because it is encoded by a dysfunctional operon. In this study, we demonstrated that the operon regulator AlkR is functional, can be induced by alkanes of various lengths, and does not suffer from product inhibition. Additionally, we identified a transposon insertion in the CYP153A operon. When the transposon was removed, the expression of the operon genes could be induced by alkanes, and the alkanes could then be oxyfunctionalized by the resulting proteins. To increase the accessibility of medium- and long-chain alkanes, we coexpressed a tunable alkane facilitator (AlkL) from GPo1. Using a recombinant strain, we produced 1.5 g/liter 1-dodecanol in 20 h and 2 g/liter 1-tetradecanol in 50 h by adding dodecane and tetradecane, respectively. Furthermore, in 68 h, we generated 3.76 g/liter of 1,12-dodecanediol by adding a dodecane-1-dodecanol substrate mixture. This study reports a very efficient method of producing C/C alkanols and C 1,12-alkanediol by whole-cell biotransformation. To produce terminally hydroxylated medium- to long-chain alkane compounds by whole-cell biotransformation, substrate permeability, enzymatic activity, and the control of overoxidability should be considered. Due to difficulties in production, small amounts of 1-dodecanol, 1-tetradecanol, and 1,12-dodecanediol are typically produced. In this study, we identified an alkane-inducible monooxygenase operon that can efficiently catalyze the conversion of alkane to 1-alkanol with no detection of the overoxidation product. By coexpressing an alkane membrane facilitator, high levels of 1-dodecanol, 1-tetradecanol, and 1,12-dodecanediol could be generated. This study is significant for the bioproduction of medium- and long-chain 1-alkanol and α,ω-alkanediols.

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Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides

Cited by 77 publications

References 30 publications

Identification of Key Residues for Enzymatic Carboxylate Reduction

Identification of Key Residues for Enzymatic Carboxylate Reduction

The Broad Aryl Acid Specificity of the Amide Bond Synthetase McbA Suggests Potential for the Biocatalytic Synthesis of Amides

Production of 1-Dodecanol, 1-Tetradecanol, and 1,12-Dodecanediol through Whole-Cell Biotransformation in Escherichia coli

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