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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

Superposition of the active site regions in AxDTA and the serine dehydratase from chicken kidney [37].Amino acids are shown as gray (AxDTA) and pink (PDB-entry: 3ANU) sticks. The cofactors are shown in yellow and the metal ions are shown as gray and pink spheres. Metal coordination is indicated by gray (AxDTA) and pink (3ANU) dashed lines. Water molecules are shown as small red (AxDTA) and dark red (3ANU) spheres.
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pone.0124056.g006: Superposition of the active site regions in AxDTA and the serine dehydratase from chicken kidney [37].Amino acids are shown as gray (AxDTA) and pink (PDB-entry: 3ANU) sticks. The cofactors are shown in yellow and the metal ions are shown as gray and pink spheres. Metal coordination is indicated by gray (AxDTA) and pink (3ANU) dashed lines. Water molecules are shown as small red (AxDTA) and dark red (3ANU) spheres.

Mentions: The putative mechanism involves the manganese ion as a crucial Lewis acid, which is in line with previous findings showing that divalent metal ions are important for enzyme activity [7]. In contrast to well-studied PLP-dependent enzyme reactions assisted by monovalent ions, where the metal ion can be either directly involved in catalysis or via allosteric effects [35], detailed analyses of PLP-dependent reactions involving divalent ions have been described to a lesser extent [7,31,36,37]. A search in the PDB using the program Relibase+ [38] yielded only two structures, which resemble AxDTA in terms of metal binding architecture and the relative arrangement of the metal binding site and the PLP cofactor. One of them is an enzyme from Idiomarina loihiensis annotated as “predicted amino acid aldolase or racemase”. Its structure (PDB entry 3LLX) was determined by the Joint Center for Structural Genomics (JCSG), but no paper has so far been published. It should be noted that this structure was used as the molecular replacement search template in the structure determination of AxDTA. The second enzyme is a D-serine dehydratase from chicken kidneys (PDB-entry 3ANU) [37]. In both cases, a zinc ion is bound at the same position as the manganese in AxDTA (Fig 6). In the case of the serine dehydratase it has been reported that the zinc ion can be replaced by manganese without loss of activity [37]. Based on crystal structures and molecular modeling a catalytic mechanism has been proposed for this enzyme, which is very similar to our proposed mechanism for AxDTA (Fig 5) and also involves activation of the substrate OH-group by coordinating to the metal ion [37]. Metal coordination in the dehydratase and the predicted aldolase/racemase, however, is achieved by a histidine and a cysteine instead of a histidine and an aspartate in AxDTA (Fig 6). In addition, the potential base His193 in AxDTA is not present in the other two enzymes.


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 the active site regions in AxDTA and the serine dehydratase from chicken kidney [37].Amino acids are shown as gray (AxDTA) and pink (PDB-entry: 3ANU) sticks. The cofactors are shown in yellow and the metal ions are shown as gray and pink spheres. Metal coordination is indicated by gray (AxDTA) and pink (3ANU) dashed lines. Water molecules are shown as small red (AxDTA) and dark red (3ANU) spheres.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124056.g006: Superposition of the active site regions in AxDTA and the serine dehydratase from chicken kidney [37].Amino acids are shown as gray (AxDTA) and pink (PDB-entry: 3ANU) sticks. The cofactors are shown in yellow and the metal ions are shown as gray and pink spheres. Metal coordination is indicated by gray (AxDTA) and pink (3ANU) dashed lines. Water molecules are shown as small red (AxDTA) and dark red (3ANU) spheres.
Mentions: The putative mechanism involves the manganese ion as a crucial Lewis acid, which is in line with previous findings showing that divalent metal ions are important for enzyme activity [7]. In contrast to well-studied PLP-dependent enzyme reactions assisted by monovalent ions, where the metal ion can be either directly involved in catalysis or via allosteric effects [35], detailed analyses of PLP-dependent reactions involving divalent ions have been described to a lesser extent [7,31,36,37]. A search in the PDB using the program Relibase+ [38] yielded only two structures, which resemble AxDTA in terms of metal binding architecture and the relative arrangement of the metal binding site and the PLP cofactor. One of them is an enzyme from Idiomarina loihiensis annotated as “predicted amino acid aldolase or racemase”. Its structure (PDB entry 3LLX) was determined by the Joint Center for Structural Genomics (JCSG), but no paper has so far been published. It should be noted that this structure was used as the molecular replacement search template in the structure determination of AxDTA. The second enzyme is a D-serine dehydratase from chicken kidneys (PDB-entry 3ANU) [37]. In both cases, a zinc ion is bound at the same position as the manganese in AxDTA (Fig 6). In the case of the serine dehydratase it has been reported that the zinc ion can be replaced by manganese without loss of activity [37]. Based on crystal structures and molecular modeling a catalytic mechanism has been proposed for this enzyme, which is very similar to our proposed mechanism for AxDTA (Fig 5) and also involves activation of the substrate OH-group by coordinating to the metal ion [37]. Metal coordination in the dehydratase and the predicted aldolase/racemase, however, is achieved by a histidine and a cysteine instead of a histidine and an aspartate in AxDTA (Fig 6). In addition, the potential base His193 in AxDTA is not present in the other two enzymes.

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