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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.

No MeSH data available.


Related in: MedlinePlus

Stereo representation of the modeled complex of AxDTA with (2R,3S)-phenylserine.Amino acid residues are shown as gray sticks. The external aldimine intermediate is shown in yellow. The manganese ion is depicted as a magenta sphere. Potential hydrogen bonds as well as the metal coordination are indicated by light blue, dashed lines.
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pone.0124056.g004: Stereo representation of the modeled complex of AxDTA with (2R,3S)-phenylserine.Amino acid residues are shown as gray sticks. The external aldimine intermediate is shown in yellow. The manganese ion is depicted as a magenta sphere. Potential hydrogen bonds as well as the metal coordination are indicated by light blue, dashed lines.

Mentions: The resulting complex structure was further geometry optimized and is shown in Fig 4. In this structure the carboxylate of the substrate forms a salt bridge with the guanidinium group of Arg157 and is hydrogen bonded to the main-chain amide group of Asp321. The Cβ-hydroxy-group is coordinating the manganese ion with a Mn-O distance of 2.3 Å. In that position it replaces a water molecule that is bound to the ion in the crystal structure of AxDTA. Another water molecule forms a bridge between the OH-group of the substrate and the imidazole ring of His193. The phenyl ring is located in the entrance funnel to the active site and is pointing towards the exterior of the enzyme.


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)

Stereo representation of the modeled complex of AxDTA with (2R,3S)-phenylserine.Amino acid residues are shown as gray sticks. The external aldimine intermediate is shown in yellow. The manganese ion is depicted as a magenta sphere. Potential hydrogen bonds as well as the metal coordination are indicated by light blue, dashed lines.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124056.g004: Stereo representation of the modeled complex of AxDTA with (2R,3S)-phenylserine.Amino acid residues are shown as gray sticks. The external aldimine intermediate is shown in yellow. The manganese ion is depicted as a magenta sphere. Potential hydrogen bonds as well as the metal coordination are indicated by light blue, dashed lines.
Mentions: The resulting complex structure was further geometry optimized and is shown in Fig 4. In this structure the carboxylate of the substrate forms a salt bridge with the guanidinium group of Arg157 and is hydrogen bonded to the main-chain amide group of Asp321. The Cβ-hydroxy-group is coordinating the manganese ion with a Mn-O distance of 2.3 Å. In that position it replaces a water molecule that is bound to the ion in the crystal structure of AxDTA. Another water molecule forms a bridge between the OH-group of the substrate and the imidazole ring of His193. The phenyl ring is located in the entrance funnel to the active site and is pointing towards the exterior of the enzyme.

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