Structural determinants for the non-canonical substrate specificity of the ω-transaminase from paracoccus denitrificans

Eul Soo Park, Sae Rom Park, Sang Woo Han, Joo Young Dong, jong shik shin

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Substrate binding pockets of ω-transaminase (ω-TA) consist of a large (L) pocket capable of dual recognition of hydrophobic and carboxyl substituents, and a small (S) pocket displaying a strict steric constraint that permits entry of a substituent no larger than an ethyl group. Despite the unique catalytic utility of ω-TA enabling asymmetric reductive amination of carbonyl compounds, the severe size exclusion occurring in the S pocket has limited synthetic applications of ω-TA to access structurally diverse chiral amines and amino acids. Here we report the first example of an ω-TA whose S pocket shows a non-canonical steric constraint and readily accommodates up to an n-butyl substituent. The relaxed substrate specificity of the (S)-selective ω-TA, cloned from Paracoccus denitrificans (PDTA), afforded efficient asymmetric syntheses of unnatural amino acids carrying long alkyl side chains such as lnorvaline and l-norleucine. Molecular modeling using the recently released X-ray structure of PDTA could pinpoint an exact location of the S pocket which had remained dubious. Entry of a hydrophobic substituent in the L pocket was found to have the S pocket accept up to an ethyl substituent, reminiscent of the canonical steric constraint. In contrast, binding of a carboxyl group to the L pocket induced a slight movement of V153 away from the small-pocketforming residues. The resulting structural change elicited excavation of the S pocket, leading to formation of a narrow tunnel-like structure allowing accommodation of linear alkyl groups of carboxylatebearing substrates. To verify the active site model, we introduced site-directed mutagenesis to six active site residues and examined whether the point mutations alleviated the steric constraint in the S pocket. Consistent with the molecular modeling results, the V153A variant assumed an elongated S pocket and accepted even an n-hexyl substituent. Our findings provide precise structural information on substrate binding to the active site of ω-TA, which is expected to benefit rational redesign of substrate specificity of ω-TA.

Original languageEnglish
Pages (from-to)212-220
Number of pages9
JournalAdvanced Synthesis and Catalysis
Volume356
Issue number1
DOIs
Publication statusPublished - 2014 Jan 1

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Transaminases
Substrates
Molecular modeling
Amino acids
Norleucine
Amination
Amino Acids
Carbonyl compounds
Mutagenesis
Excavation
Amines
Tunnels
X rays

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Organic Chemistry

Cite this

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abstract = "Substrate binding pockets of ω-transaminase (ω-TA) consist of a large (L) pocket capable of dual recognition of hydrophobic and carboxyl substituents, and a small (S) pocket displaying a strict steric constraint that permits entry of a substituent no larger than an ethyl group. Despite the unique catalytic utility of ω-TA enabling asymmetric reductive amination of carbonyl compounds, the severe size exclusion occurring in the S pocket has limited synthetic applications of ω-TA to access structurally diverse chiral amines and amino acids. Here we report the first example of an ω-TA whose S pocket shows a non-canonical steric constraint and readily accommodates up to an n-butyl substituent. The relaxed substrate specificity of the (S)-selective ω-TA, cloned from Paracoccus denitrificans (PDTA), afforded efficient asymmetric syntheses of unnatural amino acids carrying long alkyl side chains such as lnorvaline and l-norleucine. Molecular modeling using the recently released X-ray structure of PDTA could pinpoint an exact location of the S pocket which had remained dubious. Entry of a hydrophobic substituent in the L pocket was found to have the S pocket accept up to an ethyl substituent, reminiscent of the canonical steric constraint. In contrast, binding of a carboxyl group to the L pocket induced a slight movement of V153 away from the small-pocketforming residues. The resulting structural change elicited excavation of the S pocket, leading to formation of a narrow tunnel-like structure allowing accommodation of linear alkyl groups of carboxylatebearing substrates. To verify the active site model, we introduced site-directed mutagenesis to six active site residues and examined whether the point mutations alleviated the steric constraint in the S pocket. Consistent with the molecular modeling results, the V153A variant assumed an elongated S pocket and accepted even an n-hexyl substituent. Our findings provide precise structural information on substrate binding to the active site of ω-TA, which is expected to benefit rational redesign of substrate specificity of ω-TA.",
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Structural determinants for the non-canonical substrate specificity of the ω-transaminase from paracoccus denitrificans. / Park, Eul Soo; Park, Sae Rom; Han, Sang Woo; Dong, Joo Young; shin, jong shik.

In: Advanced Synthesis and Catalysis, Vol. 356, No. 1, 01.01.2014, p. 212-220.

Research output: Contribution to journalArticle

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