Key Amino Acid Residues for Reversed or Improved Enantiospecificity of an ω‐Transaminase

Humble, Maria Svedendahl; Cassimjee, Karim Engelmark; Abedi, Vahak; Federsel, Hans‐Jürgen; Berglund, Per

doi:10.1002/cctc.201100487

Cited by 47 publications

(51 citation statements)

References 54 publications

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“…Humble et al . previously observed that Y153K decreased the enzyme activity, and attributed this to the loss of the edge‐on interaction of the Y153 phenyl ring with the pyridine ring of PLP, as well as loss of the hydrogen bond via water to the phosphate moiety of PLP. However, mutations to almost any other residue than charged variants, and notably Y153F, remove the hydrogen bond interaction and yet lead to increased activity towards serine.…”

Section: Resultsmentioning

confidence: 88%

Single active‐site mutants are sufficient to enhance serine:pyruvate α‐transaminase activity in an ω‐transaminase

et al. 2015

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We have analyzed the natural evolution of transaminase structure and sequence between an a-transaminase serine-pyruvate aminotransferase and an x-transaminase from Chromobacterium violaceum with < 20% sequence identity, and identified the active-site regions that are least conserved structurally. We also show that these structural changes correlate strongly with transaminase substrate specificity during evolution and therefore might normally be presumed to be essential determinants of substrate specificity. However, key residues are often conserved spatially during evolution and yet originate from within a different region of the sequence via structural reorganizations. In the present study, we also show that a-transaminase-type serine-pyruvate aminotransferase activity can be engineered into the CV2025 x-transaminase scaffold with any one of many possible single-point mutations at three key positions, without the requirement for significant backbone remodeling, or repositioning of the residue from a different region of sequence. This finding has significant implications for enzyme redesign in which solutions to substrate specificity changes may be found more efficiently than is achieved by engineering in all sequence and structure determinants identified by correlation to substrate specificity.

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Section: Resultsmentioning

confidence: 88%

Single active‐site mutants are sufficient to enhance serine:pyruvate α‐transaminase activity in an ω‐transaminase

et al. 2015

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“…Four positions in this “partial signature”—Trp57, Phe85*, Tyr150, and Ala228 were previously studied by site‐directed mutagenesis of VF ω‐AT 82 and CV ω‐AT83 in attempts to redesign the substrate specificity and to understand the enantioselectivity preference of ω‐ATs. The mutation Trp57Gly in VF ω‐AT resulted in improved catalytic activities for aromatic and aliphatic amines without compromising enantioselectivity.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of the ω‐aminotransferase from Paracoccus denitrificans and its phylogenetic relationship with other class III amino‐ transferases that have biotechnological potential

Rausch¹,

Lerchner²,

Schiefner³

et al. 2013

Proteins

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Apart from their crucial role in metabolism, pyridoxal 5'-phosphate (PLP)-dependent aminotransferases (ATs) constitute a class of enzymes with increasing application in industrial biotechnology. To provide better insight into the structure-function relationships of ATs with biotechnological potential we performed a fundamental bioinformatics analysis of 330 representative sequences of pro- and eukaryotic Class III ATs using a structure-guided approach. The calculated phylogenetic maximum likelihood tree revealed six distinct clades of which the first segregates with a very high bootstrap value of 92%. Most enzymes in this first clade have been functionally well characterized, whereas knowledge about the natural functions and substrates of enzymes in the other branches is sparse. Notably, in those clades 2-6 members of the peculiar class of ω-ATs prevail, many of which have proven useful for the preparation of chiral amines or artificial amino acids. One representative is the ω-AT from Paracoccus denitrificans (PD ω-AT) which catalyzes, for example, the transamination in a novel biocatalytic process for the production of L-homoalanine from L-threonine. To gain structural insight into this important enzyme, its X-ray analysis was carried out at a resolution of 2.6 Å, including the covalently bound PLP as well as 5-aminopentanoate as a putative amino donor substrate. On the basis of this crystal structure in conjunction with our phylogenetic analysis, we have identified a generic set of active site residues of ω-ATs that are associated with a strong preference for aromatic substrates, thus guiding the discovery of novel promising enzymes for the biotechnological production of corresponding chiral amines.

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“…In subunit B, the internal aldimine was observed similar to the complex found in subunit B in the 6‐AHA binding study. The structures determined of Pj AT resemble most the structures of Oa AT with PMP bound (http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5GHF), of Cv AT with PLP bound (PDB http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4A6T) and with gabaculine‐PLP bound ( m ‐carboxyphenyl pyridoxamine phosphate) (http://www.rcsb.org/pdb/search/structidSearch.do?structureId=4BA5) .…”

Section: Resultsmentioning

confidence: 80%

“…Displayed are the sequences of Pj AT from Pseudomonas jessenii, Oa AT from Ochrobactrum anthropi ( 5GHF, ), a putative AT from Mesorhizobium loti maff303099 ( Ml AT , 3GJU, JCSG ), ω‐ AT from Paracoccus denitrificans ( Pd AT , 4GRX, ), Vf AT from Vibrio fluvialis ( 4E3Q, ), AT from Silicibacter pomeroyi ( Sp AT , 3 HMU , NYSGXRC ), a putative AT from Silicibacter sp. TM 1040 ( Si AT , 3 FCR , JCSG ), and Cv AT from Chromobacterium violaceum ( 4A6R, 4A6T, 4BA5 ). The sequences have more than 40% identity.…”

Section: Resultsmentioning

confidence: 99%

“…aeruginosa transaminase . However, conformational changes upon binding of a phosphate or phosphate mimic of several loops involved in structuring the active site may be possible in ATs, like displayed in Cv AT . Both Cv AT and Vf AT have phenylalanine side chains pointing toward the conserved arginine (Arg417 in Pj AT), and restricting space at the active site entrance (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Biochemical properties of a Pseudomonas aminotransferase involved in caprolactam metabolism

et al. 2019

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The biodegradation of the nylon‐6 precursor caprolactam by a strain of Pseudomonas jessenii proceeds via ATP‐dependent hydrolytic ring opening to 6‐aminohexanoate. This non‐natural ω‐amino acid is converted to 6‐oxohexanoic acid by an aminotransferase (PjAT) belonging to the fold type I pyridoxal 5′‐phosphate (PLP) enzymes. To understand the structural basis of 6‐aminohexanoatate conversion, we solved different crystal structures and determined the substrate scope with a range of aliphatic and aromatic amines. Comparison with the homologous aminotransferases from Chromobacterium violaceum (CvAT) and Vibrio fluvialis (VfAT) showed that the PjAT enzyme has the lowest KM values (highest affinity) and highest specificity constant (kcat/KM) with the caprolactam degradation intermediates 6‐aminohexanoate and 6‐oxohexanoic acid, in accordance with its proposed in vivo function. Five distinct three‐dimensional structures of PjAT were solved by protein crystallography. The structure of the aldimine intermediate formed from 6‐aminohexanoate and the PLP cofactor revealed the presence of a narrow hydrophobic substrate‐binding tunnel leading to the cofactor and covered by a flexible arginine, which explains the high activity and selectivity of the PjAT with 6‐aminohexanoate. The results suggest that the degradation pathway for caprolactam has recruited an aminotransferase that is well adapted to 6‐aminohexanoate degradation. Database The atomic coordinates and structure factors P. jessenii 6‐aminohexanoate aminotransferase have been deposited in the PDB as entries http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G4B (E∙succinate complex), http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G4C (E∙phosphate complex), http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G4D (E∙PLP complex), http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G4E (E∙PLP‐6‐aminohexanoate intermediate), and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G4F (E∙PMP complex).

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Key Amino Acid Residues for Reversed or Improved Enantiospecificity of an ω‐Transaminase

Cited by 47 publications

References 54 publications

Single active‐site mutants are sufficient to enhance serine:pyruvate α‐transaminase activity in an ω‐transaminase

Single active‐site mutants are sufficient to enhance serine:pyruvate α‐transaminase activity in an ω‐transaminase

Crystal structure of the ω‐aminotransferase from Paracoccus denitrificans and its phylogenetic relationship with other class III amino‐ transferases that have biotechnological potential

Biochemical properties of a Pseudomonas aminotransferase involved in caprolactam metabolism

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