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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

Structure of the active site and the metal binding sites of AxDTA.(A) Close-up view of the region around the PLP-cofactor (shown in yellow). Amino acid residues are shown as gray sticks. The manganese ion is depicted as a magenta sphere. Water molecules are shown as small red spheres. Potential hydrogen bonds and the metal coordination are indicated by light blue, dashed lines. (B) Close-up view of the sodium ion (green sphere) binding site. Residues coordinating the Na-ion are shown as gray sticks, metal bound water molecules are shown as small red spheres. Metal coordination is indicated by light blue, dashed lines.
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pone.0124056.g003: Structure of the active site and the metal binding sites of AxDTA.(A) Close-up view of the region around the PLP-cofactor (shown in yellow). Amino acid residues are shown as gray sticks. The manganese ion is depicted as a magenta sphere. Water molecules are shown as small red spheres. Potential hydrogen bonds and the metal coordination are indicated by light blue, dashed lines. (B) Close-up view of the sodium ion (green sphere) binding site. Residues coordinating the Na-ion are shown as gray sticks, metal bound water molecules are shown as small red spheres. Metal coordination is indicated by light blue, dashed lines.

Mentions: The active site of AxDTA is mainly built up from residues of the alanine racemase-like domain (Fig 3A). The smaller β-domain of the second protomer contributes residues from two loop regions (residues 295–298 and 319–323). Clear, residual electron density in the active site of both protomers was modeled by a molecule of pyridoxal phosphate (PLP) in two alternate conformations: covalently linked to Lys59 (occupancy ~40%) and non-covalently bound (occupancy ~60%). In general PLP binds covalently to an active-site lysine in the resting state of the enzyme (referred to as the internal aldimine) [1]. In both cases, the pyridine ring of PLP forms π-stacking interactions with Tyr187 (distance ~3.7 Å). The pyridine nitrogen atom forms a hydrogen bond to Gln249. The phosphate group of the cofactor interacts with the hydroxyl groups of Thr233, Ser252 and Tyr260 as well as with main chain amide groups of Thr233 and Gly251. The hydroxyl group at the pyridine ring of PLP forms hydrogen bonds with the side chain amide of Gln81 and the guanidinium group of Arg157.


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)

Structure of the active site and the metal binding sites of AxDTA.(A) Close-up view of the region around the PLP-cofactor (shown in yellow). Amino acid residues are shown as gray sticks. The manganese ion is depicted as a magenta sphere. Water molecules are shown as small red spheres. Potential hydrogen bonds and the metal coordination are indicated by light blue, dashed lines. (B) Close-up view of the sodium ion (green sphere) binding site. Residues coordinating the Na-ion are shown as gray sticks, metal bound water molecules are shown as small red spheres. Metal coordination is indicated by light blue, dashed lines.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124056.g003: Structure of the active site and the metal binding sites of AxDTA.(A) Close-up view of the region around the PLP-cofactor (shown in yellow). Amino acid residues are shown as gray sticks. The manganese ion is depicted as a magenta sphere. Water molecules are shown as small red spheres. Potential hydrogen bonds and the metal coordination are indicated by light blue, dashed lines. (B) Close-up view of the sodium ion (green sphere) binding site. Residues coordinating the Na-ion are shown as gray sticks, metal bound water molecules are shown as small red spheres. Metal coordination is indicated by light blue, dashed lines.
Mentions: The active site of AxDTA is mainly built up from residues of the alanine racemase-like domain (Fig 3A). The smaller β-domain of the second protomer contributes residues from two loop regions (residues 295–298 and 319–323). Clear, residual electron density in the active site of both protomers was modeled by a molecule of pyridoxal phosphate (PLP) in two alternate conformations: covalently linked to Lys59 (occupancy ~40%) and non-covalently bound (occupancy ~60%). In general PLP binds covalently to an active-site lysine in the resting state of the enzyme (referred to as the internal aldimine) [1]. In both cases, the pyridine ring of PLP forms π-stacking interactions with Tyr187 (distance ~3.7 Å). The pyridine nitrogen atom forms a hydrogen bond to Gln249. The phosphate group of the cofactor interacts with the hydroxyl groups of Thr233, Ser252 and Tyr260 as well as with main chain amide groups of Thr233 and Gly251. The hydroxyl group at the pyridine ring of PLP forms hydrogen bonds with the side chain amide of Gln81 and the guanidinium group of Arg157.

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