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Structure of Arabidopsis thaliana 5-methylthioribose kinase reveals a more occluded active site than its bacterial homolog.

Ku SY, Cornell KA, Howell PL - BMC Struct. Biol. (2007)

Bottom Line: The ATPgammaS analog was hydrolyzed during the co-crystallization procedure, resulting in ADP in the active site.The structure of A. thaliana MTR kinase provides a template for structure-based design of agrochemicals, particularly herbicides whose effectiveness could be regulated by nutrient levels.Features of the MTR binding site offer an opportunity for a simple organic salt of an MTR analog to specifically inhibit MTR kinase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Molecular Structure and Function, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, CANADA. syku@sickkids.ca

ABSTRACT

Background: Metabolic variations exist between the methionine salvage pathway of humans and a number of plants and microbial pathogens. 5-Methylthioribose (MTR) kinase is a key enzyme required for methionine salvage in plants and many bacteria. The absence of a mammalian homolog suggests that MTR kinase is a good target for the design of specific herbicides or antibiotics.

Results: The structure of Arabidopsis thaliana MTR kinase co-crystallized with ATPgammaS and MTR has been determined at 1.9 A resolution. The structure is similar to B. subtilis MTR kinase and has the same protein kinase fold observed in other evolutionarily related protein kinase-like phosphotransferases. The active site is comparable between the two enzymes with the DXE-motif coordinating the nucleotide-Mg, the D238 of the HGD catalytic loop polarizing the MTR O1 oxygen, and the RR-motif interacting with the substrate MTR. Unlike its bacterial homolog, however, the Gly-rich loop (G-loop) of A. thaliana MTR kinase has an extended conformation, which shields most of the active site from solvent, a feature that resembles eukaryotic protein kinases more than the bacterial enzyme. The G- and W-loops of A. thaliana and B. subtilis MTR kinase adopt different conformations despite high sequence similarity. The ATPgammaS analog was hydrolyzed during the co-crystallization procedure, resulting in ADP in the active site. This suggests that the A. thaliana enzyme, like its bacterial homolog, may have significant ATPase activity in the absence of MTR.

Conclusion: The structure of A. thaliana MTR kinase provides a template for structure-based design of agrochemicals, particularly herbicides whose effectiveness could be regulated by nutrient levels. Features of the MTR binding site offer an opportunity for a simple organic salt of an MTR analog to specifically inhibit MTR kinase.

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Comparison of the nucleotide binding pockets and loop conformations. Stereo surface representation of the A. thaliana MTR kinase ADP-MTR complex and B. subtilis MTR kinase AMPPCP-MTR complex are shown in panel (a) and (c), respectively with the G- and W-loop coloured in green and the ligands shown as purple sticks. Stereo cartoon representations of the four functionally important loops, the G-loop, the W-loop, the Mg-binding DXE-motif, and the HGD catalytic loop, found in the A. thaliana and B. subtilis enzymes are shown in (b) and (d), respectively. Substrates and important residues discussed in the text are shown in stick presentation with the same colour scheme as in Figure 4b. Residues labelled with an asterisk indicate that disordered side chains are observed in at least one subunit of all known structures of the B. subtilis enzyme. This figure was prepared using PyMOL [51].
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Figure 5: Comparison of the nucleotide binding pockets and loop conformations. Stereo surface representation of the A. thaliana MTR kinase ADP-MTR complex and B. subtilis MTR kinase AMPPCP-MTR complex are shown in panel (a) and (c), respectively with the G- and W-loop coloured in green and the ligands shown as purple sticks. Stereo cartoon representations of the four functionally important loops, the G-loop, the W-loop, the Mg-binding DXE-motif, and the HGD catalytic loop, found in the A. thaliana and B. subtilis enzymes are shown in (b) and (d), respectively. Substrates and important residues discussed in the text are shown in stick presentation with the same colour scheme as in Figure 4b. Residues labelled with an asterisk indicate that disordered side chains are observed in at least one subunit of all known structures of the B. subtilis enzyme. This figure was prepared using PyMOL [51].

Mentions: In A. thaliana MTR kinase, the G-loop plays a similar role in anchoring and coordinating the bound nucleotide. Residue D44 of the G-loop, although not absolutely conserved, makes a ~2.6 Å hydrogen bond to the nucleotide's O3' ribosyl oxygen, while the backbone and terminal amide nitrogen's of N48 interact with the β-phosphoryl oxygens. The G-loop of A. thaliana MTR kinase has an extended conformation, with interactions formed between N46 and the O4 oxygen of MTR (Fig. 4b and 5a). This extended G-loop conformation is different from that observed in the bacterial MTR kinase, as the equivalent residue in the B. subtilis enzyme, N42, is over 10 Å away from the O4 oxygen of MTR (Fig. 5d). In most of the bacterial structures determined to date [26], the G-loop is disordered, and in no case does it interact with MTR. A consequence of this extended G-loop in the Arabidopsis enzyme is that it shields the active site from solvent (Fig. 5a), a feature that more closely resembles a eukaryotic protein kinase active site than that of the B. subtilis MTR kinase (Fig. 5c). Since the G-loop appears to deny the entry and/or exit of nucleotides, conformational changes – loop movement at a minimum – will need to occur to allow nucleotide entry to the apo-form the enzyme.


Structure of Arabidopsis thaliana 5-methylthioribose kinase reveals a more occluded active site than its bacterial homolog.

Ku SY, Cornell KA, Howell PL - BMC Struct. Biol. (2007)

Comparison of the nucleotide binding pockets and loop conformations. Stereo surface representation of the A. thaliana MTR kinase ADP-MTR complex and B. subtilis MTR kinase AMPPCP-MTR complex are shown in panel (a) and (c), respectively with the G- and W-loop coloured in green and the ligands shown as purple sticks. Stereo cartoon representations of the four functionally important loops, the G-loop, the W-loop, the Mg-binding DXE-motif, and the HGD catalytic loop, found in the A. thaliana and B. subtilis enzymes are shown in (b) and (d), respectively. Substrates and important residues discussed in the text are shown in stick presentation with the same colour scheme as in Figure 4b. Residues labelled with an asterisk indicate that disordered side chains are observed in at least one subunit of all known structures of the B. subtilis enzyme. This figure was prepared using PyMOL [51].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2194712&req=5

Figure 5: Comparison of the nucleotide binding pockets and loop conformations. Stereo surface representation of the A. thaliana MTR kinase ADP-MTR complex and B. subtilis MTR kinase AMPPCP-MTR complex are shown in panel (a) and (c), respectively with the G- and W-loop coloured in green and the ligands shown as purple sticks. Stereo cartoon representations of the four functionally important loops, the G-loop, the W-loop, the Mg-binding DXE-motif, and the HGD catalytic loop, found in the A. thaliana and B. subtilis enzymes are shown in (b) and (d), respectively. Substrates and important residues discussed in the text are shown in stick presentation with the same colour scheme as in Figure 4b. Residues labelled with an asterisk indicate that disordered side chains are observed in at least one subunit of all known structures of the B. subtilis enzyme. This figure was prepared using PyMOL [51].
Mentions: In A. thaliana MTR kinase, the G-loop plays a similar role in anchoring and coordinating the bound nucleotide. Residue D44 of the G-loop, although not absolutely conserved, makes a ~2.6 Å hydrogen bond to the nucleotide's O3' ribosyl oxygen, while the backbone and terminal amide nitrogen's of N48 interact with the β-phosphoryl oxygens. The G-loop of A. thaliana MTR kinase has an extended conformation, with interactions formed between N46 and the O4 oxygen of MTR (Fig. 4b and 5a). This extended G-loop conformation is different from that observed in the bacterial MTR kinase, as the equivalent residue in the B. subtilis enzyme, N42, is over 10 Å away from the O4 oxygen of MTR (Fig. 5d). In most of the bacterial structures determined to date [26], the G-loop is disordered, and in no case does it interact with MTR. A consequence of this extended G-loop in the Arabidopsis enzyme is that it shields the active site from solvent (Fig. 5a), a feature that more closely resembles a eukaryotic protein kinase active site than that of the B. subtilis MTR kinase (Fig. 5c). Since the G-loop appears to deny the entry and/or exit of nucleotides, conformational changes – loop movement at a minimum – will need to occur to allow nucleotide entry to the apo-form the enzyme.

Bottom Line: The ATPgammaS analog was hydrolyzed during the co-crystallization procedure, resulting in ADP in the active site.The structure of A. thaliana MTR kinase provides a template for structure-based design of agrochemicals, particularly herbicides whose effectiveness could be regulated by nutrient levels.Features of the MTR binding site offer an opportunity for a simple organic salt of an MTR analog to specifically inhibit MTR kinase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Molecular Structure and Function, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, CANADA. syku@sickkids.ca

ABSTRACT

Background: Metabolic variations exist between the methionine salvage pathway of humans and a number of plants and microbial pathogens. 5-Methylthioribose (MTR) kinase is a key enzyme required for methionine salvage in plants and many bacteria. The absence of a mammalian homolog suggests that MTR kinase is a good target for the design of specific herbicides or antibiotics.

Results: The structure of Arabidopsis thaliana MTR kinase co-crystallized with ATPgammaS and MTR has been determined at 1.9 A resolution. The structure is similar to B. subtilis MTR kinase and has the same protein kinase fold observed in other evolutionarily related protein kinase-like phosphotransferases. The active site is comparable between the two enzymes with the DXE-motif coordinating the nucleotide-Mg, the D238 of the HGD catalytic loop polarizing the MTR O1 oxygen, and the RR-motif interacting with the substrate MTR. Unlike its bacterial homolog, however, the Gly-rich loop (G-loop) of A. thaliana MTR kinase has an extended conformation, which shields most of the active site from solvent, a feature that resembles eukaryotic protein kinases more than the bacterial enzyme. The G- and W-loops of A. thaliana and B. subtilis MTR kinase adopt different conformations despite high sequence similarity. The ATPgammaS analog was hydrolyzed during the co-crystallization procedure, resulting in ADP in the active site. This suggests that the A. thaliana enzyme, like its bacterial homolog, may have significant ATPase activity in the absence of MTR.

Conclusion: The structure of A. thaliana MTR kinase provides a template for structure-based design of agrochemicals, particularly herbicides whose effectiveness could be regulated by nutrient levels. Features of the MTR binding site offer an opportunity for a simple organic salt of an MTR analog to specifically inhibit MTR kinase.

Show MeSH
Related in: MedlinePlus