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The crystal structure of D-threonine aldolase from Alcaligenes xylosoxidans provides insight into a metal ion assisted PLP-dependent mechanism.

Uhl MK, Oberdorfer G, Steinkellner G, Riegler-Berket L, Mink D, van Assema F, Schürmann M, Gruber K - PLoS ONE (2015)

Bottom Line: Our results underline the close relationship of DTAs and alanine racemases and allow the identification of a metal binding site close to the PLP-cofactor in the active site of the enzyme which is consistent with the previous observation that divalent cations are essential for DTA activity.The structure of AxDTA is completely different to available structures of LTAs.The enantio-complementarity of DTAs and LTAs can be explained by an approximate mirror symmetry of crucial active site residues relative to the PLP-cofactor.

View Article: PubMed Central - PubMed

Affiliation: Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010, Graz, Austria.

ABSTRACT
Threonine aldolases catalyze the pyridoxal phosphate (PLP) dependent cleavage of threonine into glycine and acetaldehyde and play a major role in the degradation of this amino acid. In nature, L- as well as D-specific enzymes have been identified, but the exact physiological function of D-threonine aldolases (DTAs) is still largely unknown. Both types of enantio-complementary enzymes have a considerable potential in biocatalysis for the stereospecific synthesis of various β-hydroxy amino acids, which are valuable building blocks for the production of pharmaceuticals. While several structures of L-threonine aldolases (LTAs) have already been determined, no structure of a DTA is available to date. Here, we report on the determination of the crystal structure of the DTA from Alcaligenes xylosoxidans (AxDTA) at 1.5 Å resolution. Our results underline the close relationship of DTAs and alanine racemases and allow the identification of a metal binding site close to the PLP-cofactor in the active site of the enzyme which is consistent with the previous observation that divalent cations are essential for DTA activity. Modeling of AxDTA substrate complexes provides a rationale for this metal dependence and indicates that binding of the β-hydroxy group of the substrate to the metal ion very likely activates this group and facilitates its deprotonation by His193. An equivalent involvement of a metal ion has been implicated in the mechanism of a serine dehydratase, which harbors a metal ion binding site in the vicinity of the PLP cofactor at the same position as in DTA. The structure of AxDTA is completely different to available structures of LTAs. The enantio-complementarity of DTAs and LTAs can be explained by an approximate mirror symmetry of crucial active site residues relative to the PLP-cofactor.

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Related in: MedlinePlus

Superposition of external aldimine structures of AxDTA and the L-threonine aldolase from Thermotoga maritima (PDB-entry: 1LW4 [5]) showing the approximate mirror symmetry of the active sites.Amino acid residues in AxDTA are shown in gray, the modeled, external aldimine in yellow. The manganese ion is depicted as a magenta sphere. Residues in the LTA-structures are shown in pink, the external aldimine in blue. Potential hydrogen bonds as well as the metal coordination (in AxDTA) are indicated as light blue, dashed lines.
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pone.0124056.g007: Superposition of external aldimine structures of AxDTA and the L-threonine aldolase from Thermotoga maritima (PDB-entry: 1LW4 [5]) showing the approximate mirror symmetry of the active sites.Amino acid residues in AxDTA are shown in gray, the modeled, external aldimine in yellow. The manganese ion is depicted as a magenta sphere. Residues in the LTA-structures are shown in pink, the external aldimine in blue. Potential hydrogen bonds as well as the metal coordination (in AxDTA) are indicated as light blue, dashed lines.

Mentions: L-specific threonine aldolases (LTAs) are more common in nature than D-specific enzymes. The overall structure of AxDTA presented here is completely different to the fold exhibited by L-threonine aldolases (e.g. the enzyme from Thermotoga maritima, PDB-entry 1LW4 [5]), which involves a 3-layer α/β-sandwich instead of an (α/β)8-barrel and is closely related to the fold of aspartate amino transferases. The superposition of the PLP-cofactor provides a rationale for the inverted enantiopreference of the two types of enzymes (Fig 7). For the LTA from Thermotoga maritima a mechanism has been proposed, in which the β-OH group of the substrate is deprotonated by an active site histidine residue [5] which is located at the si-face of the cofactor. In contrast to that, the proposed base His193 is positioned at the opposite, re-face of PLP. Thus, the cofactor can be seen as a pseudo mirror plane in the superposition (Fig 7). This situation is reminiscent of a recent study of enantio-complementary ene-reductases, where the FMN-cofactor serves as the approximate mirror plane [40]. According to a recent classification of enantio-complementarity in enzymes [41] the LTA/DTA-pair is a member of group 1, which includes enzyme pairs with different folds and mirror-image active sites.


The crystal structure of D-threonine aldolase from Alcaligenes xylosoxidans provides insight into a metal ion assisted PLP-dependent mechanism.

Uhl MK, Oberdorfer G, Steinkellner G, Riegler-Berket L, Mink D, van Assema F, Schürmann M, Gruber K - PLoS ONE (2015)

Superposition of external aldimine structures of AxDTA and the L-threonine aldolase from Thermotoga maritima (PDB-entry: 1LW4 [5]) showing the approximate mirror symmetry of the active sites.Amino acid residues in AxDTA are shown in gray, the modeled, external aldimine in yellow. The manganese ion is depicted as a magenta sphere. Residues in the LTA-structures are shown in pink, the external aldimine in blue. Potential hydrogen bonds as well as the metal coordination (in AxDTA) are indicated as light blue, dashed lines.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401734&req=5

pone.0124056.g007: Superposition of external aldimine structures of AxDTA and the L-threonine aldolase from Thermotoga maritima (PDB-entry: 1LW4 [5]) showing the approximate mirror symmetry of the active sites.Amino acid residues in AxDTA are shown in gray, the modeled, external aldimine in yellow. The manganese ion is depicted as a magenta sphere. Residues in the LTA-structures are shown in pink, the external aldimine in blue. Potential hydrogen bonds as well as the metal coordination (in AxDTA) are indicated as light blue, dashed lines.
Mentions: L-specific threonine aldolases (LTAs) are more common in nature than D-specific enzymes. The overall structure of AxDTA presented here is completely different to the fold exhibited by L-threonine aldolases (e.g. the enzyme from Thermotoga maritima, PDB-entry 1LW4 [5]), which involves a 3-layer α/β-sandwich instead of an (α/β)8-barrel and is closely related to the fold of aspartate amino transferases. The superposition of the PLP-cofactor provides a rationale for the inverted enantiopreference of the two types of enzymes (Fig 7). For the LTA from Thermotoga maritima a mechanism has been proposed, in which the β-OH group of the substrate is deprotonated by an active site histidine residue [5] which is located at the si-face of the cofactor. In contrast to that, the proposed base His193 is positioned at the opposite, re-face of PLP. Thus, the cofactor can be seen as a pseudo mirror plane in the superposition (Fig 7). This situation is reminiscent of a recent study of enantio-complementary ene-reductases, where the FMN-cofactor serves as the approximate mirror plane [40]. According to a recent classification of enantio-complementarity in enzymes [41] the LTA/DTA-pair is a member of group 1, which includes enzyme pairs with different folds and mirror-image active sites.

Bottom Line: Our results underline the close relationship of DTAs and alanine racemases and allow the identification of a metal binding site close to the PLP-cofactor in the active site of the enzyme which is consistent with the previous observation that divalent cations are essential for DTA activity.The structure of AxDTA is completely different to available structures of LTAs.The enantio-complementarity of DTAs and LTAs can be explained by an approximate mirror symmetry of crucial active site residues relative to the PLP-cofactor.

View Article: PubMed Central - PubMed

Affiliation: Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010, Graz, Austria.

ABSTRACT
Threonine aldolases catalyze the pyridoxal phosphate (PLP) dependent cleavage of threonine into glycine and acetaldehyde and play a major role in the degradation of this amino acid. In nature, L- as well as D-specific enzymes have been identified, but the exact physiological function of D-threonine aldolases (DTAs) is still largely unknown. Both types of enantio-complementary enzymes have a considerable potential in biocatalysis for the stereospecific synthesis of various β-hydroxy amino acids, which are valuable building blocks for the production of pharmaceuticals. While several structures of L-threonine aldolases (LTAs) have already been determined, no structure of a DTA is available to date. Here, we report on the determination of the crystal structure of the DTA from Alcaligenes xylosoxidans (AxDTA) at 1.5 Å resolution. Our results underline the close relationship of DTAs and alanine racemases and allow the identification of a metal binding site close to the PLP-cofactor in the active site of the enzyme which is consistent with the previous observation that divalent cations are essential for DTA activity. Modeling of AxDTA substrate complexes provides a rationale for this metal dependence and indicates that binding of the β-hydroxy group of the substrate to the metal ion very likely activates this group and facilitates its deprotonation by His193. An equivalent involvement of a metal ion has been implicated in the mechanism of a serine dehydratase, which harbors a metal ion binding site in the vicinity of the PLP cofactor at the same position as in DTA. The structure of AxDTA is completely different to available structures of LTAs. The enantio-complementarity of DTAs and LTAs can be explained by an approximate mirror symmetry of crucial active site residues relative to the PLP-cofactor.

No MeSH data available.


Related in: MedlinePlus