Structural Determinants for the Non‐Canonical Substrate Specificity of the ω‐Transaminase from Paracoccus denitrificans

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 L‐norvaline 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‐pocket‐forming 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 carboxylate‐bearing 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.magnified image

Active-Site Engineering of ω-Transaminase for Production of Unnatural Amino Acids Carrying a Side Chain Bulkier than an Ethyl Substituent

“…Besides the L57A variant, the V154A variant was the only alanine substitution variant that allowed an improvement in activity for 2-oxopentanoic acid (i.e., a 3-fold increase). This result is in line with our previous observation that an alanine substitution at the same position of the -TA from P. denitrificans achieved excavation of the small pocket (29). Site-directed mutagenesis of L57.…”

“…This leads all known -TAs to accept a limited range of ␣-keto acids, such as glyoxylic acid, pyruvic acid, and 2-oxobutyric acid. The only exception to the canonical substrate specificity to date is an (S)-selective -TA from Paracoccus denitrificans that can accommodate substituents whose bulk is up to that of an n-butyl substituent of ␣-keto acid in the small pocket (29). We demonstrated that a single point mutation in the small pocket (i.e., V153A) endowed the -TA with substantial activity toward even 2-oxooctanoic acid carrying an n-hexyl side chain (29).…”

“…The only exception to the canonical substrate specificity to date is an (S)-selective -TA from Paracoccus denitrificans that can accommodate substituents whose bulk is up to that of an n-butyl substituent of ␣-keto acid in the small pocket (29). We demonstrated that a single point mutation in the small pocket (i.e., V153A) endowed the -TA with substantial activity toward even 2-oxooctanoic acid carrying an n-hexyl side chain (29). Despite the noncanonical steric constraint of the -TA from P. denitrificans, a low level of enzyme activity for isopropylamine renders a synthetic potential of the -TA less optimal for the practical amination of the bulky keto acids (21).…”

Stereoelectronic effects in the reaction of aromatic substrates catalysed by Halomonas elongata transaminase and its mutants

“…15 The enzyme topology for this class of enzymes has been well elucidated and it can be easily described by two pockets surrounding the cofactor pyridoxal phosphate (PLP). 16,17 The large one accepts bulky groups such as aromatic rings, while the second, smaller one, accommodates short hydrophobic groups.…”

“…9 On the other hand, unfavourable steric interactions between the enzyme and the substrate may hamper the reactivity and can only be gauged after investigation of the enzyme-substrate binding mode. 10 Beside stereoelectronic effects involved in the enzymatic interactions, water solubility of hydrophobic substrates may also strongly influence the reactivity. Therefore, in attempt to characterise the electronic behaviour of the amino transferase from Halomonas elongata (HEWT), 11 we investigated the role of different substituents on aromatic ketones, aldehydes, and the corresponding (S)-amines.…”