Bypassing the requirement for aminoacyl-tRNA by a cyclodipeptide synthase enzyme

Harding, Christopher John; Sutherland, Emmajay; Hanna, Jane G; Houston, Douglas R.; Czekster, Clarissa Melo

doi:10.1039/d0cb00142b

Cited by 8 publications

(14 citation statements)

References 55 publications

(84 reference statements)

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“…Previous research from our group highlighted the use of these molecules to produce CDPs however that particular enzyme -BtCDPS -only required one aa-DBE to give a product. 15 Herein we explore the use of a combination of both DBE and tRNA to yield cyclic products.…”

Section: Incorporation Of Non-canonical Amino Acids Into Cyclic Dipeptidesmentioning

confidence: 99%

“…aa-DBE has a limited half-life, which also impacts reaction yields. 15 Mass spectrometry of BtCDPS indicated that the enzyme could form the first acyl-enzyme intermediate when a DBE substrate was used. 15 Docking simulations suggested the decreased product formation when aa-DBE is a substrate occurs due to the second substrate binding and unproductive positioning in P2, lacking essential residue interactions for the reaction to proceed.…”

Section: Cdp Formation Using a Minimal Substratementioning

confidence: 99%

“…15 Mass spectrometry of BtCDPS indicated that the enzyme could form the first acyl-enzyme intermediate when a DBE substrate was used. 15 Docking simulations suggested the decreased product formation when aa-DBE is a substrate occurs due to the second substrate binding and unproductive positioning in P2, lacking essential residue interactions for the reaction to proceed. Initially we assumed Para-CDPS and Parcu-CDPS both accepted histidine in P1 as they are the only members of the CDPS family to display affinity for this amino acid.…”

Section: Cdp Formation Using a Minimal Substratementioning

confidence: 99%

“…It has been hypothesised that P1 has more stringent binding restrictions for the first amino acid compared to the larger and shallower P2 pocket. 15 The catalytic mechanism of these enzymes was first elucidated by Sauguet et al and since then details of the reaction they catalyse have been explored using experimental and computational methods. 16,17 Briefly, the first aatRNA substrate binds and the amino acid moiety (situated in the P1 pocket) is covalently transferred to the catalytic serine forming an aminoacyl-enzyme intermediate.…”

Section: Introductionmentioning

confidence: 99%

“…11,16,[18][19][20] For this reaction to proceed, there are four essential residues needed for substrate interaction and positioning: S37 and Y202 which are strictly conserved and Y178 and E182 which are highly conserved. 17 Additionally, crystal structures from both CDPS families have been solved: AlbC; 16 RV2275; 19 YvmC 17 , and BtCDPS from the NYH sub-family, 15 and Nbra-CDPS; Rgry-CDPS; and Fdum-CDPS from XYP. 21 These structures all share a common architecture featuring a Rossmann-fold domain.…”

Section: Introductionmentioning

confidence: 99%

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Structural-based engineering expands the substrate scope of a cyclodipeptide synthase

Sutherland

Harding

Czekster

2021

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Cyclodipeptide synthases (CDPSs) are a growing family of enzymes capable of producing a large variety of cyclodipetide products using aminoacylated tRNA. Histidine-containing cyclic dipeptides have important biological activities as anticancer and neuroprotective molecules. Out of the 120 experimentally validated CDPS members, only two are known to accept histidine as a substrate. Here, we studied the activities of both Para-CDPS from Parabacteroides sp. 20_3 and Parcu-CDPS from Parcubacteria bacterium RAAC4_OD1_1 which synthesise cyclo(His-Phe) and cyclo(His-Pro) respectively. Both enzymes accepted canonical and non-canonical amino acids as substrates to generate a library of novel molecules. In order to understand the substrate selectivity of these CDPSs, the crystal structure of Parcu-CDPS was solved (alongside a number of mutants) and the role of residues important for catalysis and histidine recognition were probed using mutagenesis. Three successive generations of mutants containing both single and double residue substitutions were generated leading to a change in substrate selectivity from histidine to phenylalanine and leucine. The research detailed herein is the first instance of successful engineering of a CDPS to yield different products, paving the way to direct the promiscuity of these enzymes to produce molecules of our choosing.

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Section: Incorporation Of Non-canonical Amino Acids Into Cyclic Dipeptidesmentioning

confidence: 99%

Section: Cdp Formation Using a Minimal Substratementioning

confidence: 99%