Connecting Unexplored Protein Crystal Structures to Enzymatic Function

Steffen‐Munsberg, Fabian; Vickers, Clare; Thontowi, Ahmad; Schätzle, Sebastian; Tumlirsch, Tony; Humble, Maria Svedendahl; Land, Henrik; Berglund, Per; Bornscheuer, Uwe T.; Höhne, Matthias

doi:10.1002/cctc.201200544

Cited by 70 publications

(65 citation statements)

References 34 publications

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“…With detailed kinetic analysis and calculations of free energies the same group could show that in the ( S )-transaminase from P. denitrificans differences in kcat/Km values of different substrates arise mainly from their different binding affinity than from the catalytic turnover rate [59]. This binding mode was recently also confirmed by analysing solved structures [26], [29]. Docking of α-methylbenzylamine into the structure of an ω-amino acid:pyruvate aminotransferase (PDB ID: 3A8U) from Pseudomonas putida indicated that several aromatic amino acids (Tyr 23, Phe88*, Tyr152) in the large binding pocket establish a hydrophobic environment enabling the binding of hydrophobic substituents.…”

Section: Resultsmentioning

confidence: 80%

Crystal Structure of an (R)-Selective ω-Transaminase from Aspergillus terreus

et al. 2014

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Chiral amines are important building blocks for the synthesis of pharmaceutical products, fine chemicals, and agrochemicals. ω-Transaminases are able to directly synthesize enantiopure chiral amines by catalysing the transfer of an amino group from a primary amino donor to a carbonyl acceptor with pyridoxal 5′-phosphate (PLP) as cofactor. In nature, (S)-selective amine transaminases are more abundant than the (R)-selective enzymes, and therefore more information concerning their structures is available. Here, we present the crystal structure of an (R)-ω-transaminase from Aspergillus terreus determined by X-ray crystallography at a resolution of 1.6 Å. The structure of the protein is a homodimer that displays the typical class IV fold of PLP-dependent aminotransferases. The PLP-cofactor observed in the structure is present in two states (i) covalently bound to the active site lysine (the internal aldimine form) and (ii) as substrate/product adduct (the external aldimine form) and free lysine. Docking studies revealed that (R)-transaminases follow a dual binding mode, in which the large binding pocket can harbour the bulky substituent of the amine or ketone substrate and the α-carboxylate of pyruvate or amino acids, and the small binding pocket accommodates the smaller substituent.

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Section: Resultsmentioning

confidence: 80%

Crystal Structure of an (R)-Selective ω-Transaminase from Aspergillus terreus

et al. 2014

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“…We set out to construct a homology model of OATA to identify key active-site residues responsible for rejecting the entry of bulky substituents in the small pocket. The X-ray structures used as the templates for the homology modeling were -TAs from P. denitrificans (PDB accession number 4GRX; sequence identity, 41%) (35), C. violaceum (PDB accession number 4A6T; sequence identity, 41%) (36), M. loti (PDB accession number 3GJU; sequence identity, 43%) (37), and R. sphaeroides (PDB accession number 3I5T; sequence identity, 34%) (37). The alignment of the OATA sequence with the sequences of the template -TAs is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Using four X-ray structures of (S)-selective -TAs as the templates, an homology model of OATA was constructed by use of the Modeler module (version 9.8) of the Discovery Studio package (version 3.5.0; BIOVIA, San Diego, CA). The X-ray structures used as the templates were -TAs from P. denitrificans (PDB accession number 4GRX) (35), Chromobacterium violaceum (PDB accession number 4A6T) (36), Mesorhizobium loti (PDB accession number 3GJU) (37), and Rhodobacter sphaeroides (PDB accession number 3I5T) (37). The outwardpointing arginine of the structure with PDB accession number 4GRX was set to be conserved in the homology model, resulting in a dimeric structure of OATA in which each subunit has a different conformation of the active-site arginine.…”

Section: Chemicalsmentioning

confidence: 99%

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

Han

Park

Dong

et al. 2015

Appl Environ Microbiol

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-Transaminase (-TA) is a promising enzyme for use in the production of unnatural amino acids from keto acids using cheap amino donors such as isopropylamine. The small substrate-binding pocket of most -TAs permits entry of substituents no larger than an ethyl group, which presents a significant challenge to the preparation of structurally diverse unnatural amino acids. Here we report on the engineering of an (S)-selective -TA from Ochrobactrum anthropi (OATA) to reduce the steric constraint and thereby allow the small pocket to readily accept bulky substituents. On the basis of a docking model in which L-alanine was used as a ligand, nine active-site residues were selected for alanine scanning mutagenesis. Among the resulting variants, an L57A variant showed dramatic activity improvements in activity for ␣-keto acids and ␣-amino acids carrying substituents whose bulk is up to that of an n-butyl substituent (e.g., 48-and 56-fold increases in activity for 2-oxopentanoic acid and L-norvaline, respectively). An L57G mutation also relieved the steric constraint but did so much less than the L57A mutation did. In contrast, an L57V substitution failed to induce the improvements in activity for bulky substrates. Molecular modeling suggested that the alanine substitution of L57, located in a large pocket, induces an altered binding orientation of an ␣-carboxyl group and thereby provides more room to the small pocket. The synthetic utility of the L57A variant was demonstrated by carrying out the production of optically pure L-and D-norvaline (i.e., enantiomeric excess [ee] > 99%) by asymmetric amination of 2-oxopantanoic acid and kinetic resolution of racemic norvaline, respectively. U nnatural amino acids are widely used as essential chiral building blocks for diverse pharmaceutical drugs, agrochemicals, and chiral ligands (1-3). In contrast to natural amino acids, a fermentative method for the production of unnatural amino acids is not yet commercially available (4,5). This has driven the development of various biocatalytic approaches to afford scalable processes for preparing enantiopure unnatural amino acids. These processes include kinetic resolution of racemic amino acids using acylase (6, 7), amidase (8, 9), hydantoinase (10, 11), and amino acid oxidase (12, 13) and asymmetric reductive amination of keto acids using dehydrogenase (14, 15) and transaminase (16,17). Asymmetric amination is usually favored over kinetic resolution because the former affords a 100% yield without racemization of an unwanted enantiomer.In contrast to a mandatory requirement for the supply and regeneration of an expensive external cofactor for the dehydrogenase reactions, transaminase catalyzes the transfer of an amino group from an amino donor to an acceptor using pyridoxal 5=-phosphate (PLP) as a prosthetic group (18). Nevertheless, industrial implementation of the transaminase-mediated synthesis of unnatural amino acids has lagged behind because of low equilibrium constants, usually close to unity, for the reactions between keto acids a...

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“…Ah omology model of OATA was constructed using the Modeler module (version 9.8). X-ray structures of the four (S)-selective w-TAs from Paracoccus denitrificans (PDB ID:4 GRX), [18] Chromobacteriumv iolaceum (4A6T), [19] Mesorhizobium loti (3GJU) [20] and Rhodobacter sphaeroides (3I5T) [20] were used as templates.T he four w-TAs assumeahomodimeric structure where both active-site arginines form inward conformations (i.e., pointing away from the solvent side), except for 4GRX where one subunit harborsa no utward-pointing active-site arginine.T he outward-pointing arginine of 4GRX was set to be conserved in the homology model, leading to ad imeric structure of OATA where each subunit has ad ifferent conformationofthe active-sitearginine (i.e., an inward-pointing argininei no ne subunit and an outward-pointing argininei n the other subunit). To construct ah oloenzyme structure,t he PLP moiety was copied from 4A6T.S equence alignment for the homology modelingw as carried out using BLOSUM62 as as coring matrix with ad efault setting (À900 gap open penalty; À50 gap extension penalty).…”

Section: Molecular Modelingmentioning

confidence: 99%

Mechanism‐Guided Engineering of ω‐Transaminase to Accelerate Reductive Amination of Ketones

et al. 2015

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Asymmetricr eductivea mination of ketones using w-transaminases (w-TAs)offers apromising alternative to the chemocatalytic synthesiso f chiral amines.O ne fundamental challenge to the biocatalytic strategyi st he very low enzyme activities for most ketones compared with native substrates (i.e., <1% relativet op yruvate). Here we have demonstrated that as ingle point mutation in the active site of the (S)-selective w-TAf rom Ochrobactrum anthropi could induce ar emarkable acceleration of the amination reactionw ithout anyl oss in stereoselectivity and enzyme stability.M olecular modeling of quinonoid intermediates,a lanine scanning mutagenesis and kinetic analysis revealedthat the W58 residue acted as as tericb arrier to binding and catalytic turnover of ketone substrates. Removal of the steric strain by W58L substitution, which was selected by partials aturation mutagenesis,l ed to dramatica ctivity improvements for structurally diverse ketones (e.g.,3 40-fold increase in k cat /K M for acetophenone). TheW 58Lm utant afforded an efficient synthesis of enantiopure amines (i.e., >99% ee)u sing isopropylamine as an amino donor.

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Connecting Unexplored Protein Crystal Structures to Enzymatic Function

Cited by 70 publications

References 34 publications

Crystal Structure of an (R)-Selective ω-Transaminase from Aspergillus terreus

Crystal Structure of an (R)-Selective ω-Transaminase from Aspergillus terreus

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

Mechanism‐Guided Engineering of ω‐Transaminase to Accelerate Reductive Amination of Ketones

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