Limits...
Structural insight into how Streptomyces coelicolor maltosyl transferase GlgE binds α-maltose 1-phosphate and forms a maltosyl-enzyme intermediate.

Syson K, Stevenson CE, Rashid AM, Saalbach G, Tang M, Tuukkanen A, Svergun DI, Withers SG, Lawson DM, Bornemann S - Biochemistry (2014)

Bottom Line: The X-ray structures of α-maltose 1-phosphate bound to a D394A mutein and a β-2-deoxy-2-fluoromaltosyl-enzyme intermediate with a E423A mutein were determined.There are few examples of CAZy glycoside hydrolase family 13 members that have had their glycosyl-enzyme intermediate structures determined, and none before now have been obtained with a 2-deoxy-2-fluoro substrate analogue.The covalent modification of Asp394 was confirmed using mass spectrometry.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park , Norwich NR4 7UH, United Kingdom.

ABSTRACT
GlgE (EC 2.4.99.16) is an α-maltose 1-phosphate:(1→4)-α-d-glucan 4-α-d-maltosyltransferase of the CAZy glycoside hydrolase 13_3 family. It is the defining enzyme of a bacterial α-glucan biosynthetic pathway and is a genetically validated anti-tuberculosis target. It catalyzes the α-retaining transfer of maltosyl units from α-maltose 1-phosphate to maltooligosaccharides and is predicted to use a double-displacement mechanism. Evidence of this mechanism was obtained using a combination of site-directed mutagenesis of Streptomyces coelicolor GlgE isoform I, substrate analogues, protein crystallography, and mass spectrometry. The X-ray structures of α-maltose 1-phosphate bound to a D394A mutein and a β-2-deoxy-2-fluoromaltosyl-enzyme intermediate with a E423A mutein were determined. There are few examples of CAZy glycoside hydrolase family 13 members that have had their glycosyl-enzyme intermediate structures determined, and none before now have been obtained with a 2-deoxy-2-fluoro substrate analogue. The covalent modification of Asp394 was confirmed using mass spectrometry. A similar modification of wild-type GlgE proteins from S. coelicolor and Mycobacterium tuberculosis was also observed. Small-angle X-ray scattering of the M. tuberculosis enzyme revealed a homodimeric assembly similar to that of the S. coelicolor enzyme but with slightly differently oriented monomers. The deeper understanding of the structure-function relationships of S. coelicolor GlgE will aid the development of inhibitors of the M. tuberculosis enzyme.

Show MeSH

Related in: MedlinePlus

SAXS analysis of GlgE proteins. The uppercurves (A) show the SAXSdata and fit for the S. coelicolor GlgE protein (χ= 1.03; experimental data colored blue and the theoretical profilecolored red on the basis of its X-ray crystal structure). The lowercurves show the M. tuberculosis GlgE SAXS data andfit of the theoretical profile of the initial homology model basedon the S. coelicolor GlgE crystal structure (χ= 1.34; experimental data colored green and fit colored blue) andafter the GENCRY rigid body refinement giving a significantly betterfit particularly in the range of 0.1–0.2 Å–1 (χ = 1.09; theoretical fit colored red), consistent with abetter relative orientation of the monomers. The SAXS profiles aredisplaced along the logarithmic axis for the sake of clarity. Thehomology model of the M. tuberculosis GlgE dimer(B) based on the S. coelicolor GlgE structure before(yellow) and after (red) rigid body refinement gave a root-mean-squaredeviation of 6.8 Å. The overall orientation of the dimer is similarto that in Figure 1.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4048318&req=5

fig4: SAXS analysis of GlgE proteins. The uppercurves (A) show the SAXSdata and fit for the S. coelicolor GlgE protein (χ= 1.03; experimental data colored blue and the theoretical profilecolored red on the basis of its X-ray crystal structure). The lowercurves show the M. tuberculosis GlgE SAXS data andfit of the theoretical profile of the initial homology model basedon the S. coelicolor GlgE crystal structure (χ= 1.34; experimental data colored green and fit colored blue) andafter the GENCRY rigid body refinement giving a significantly betterfit particularly in the range of 0.1–0.2 Å–1 (χ = 1.09; theoretical fit colored red), consistent with abetter relative orientation of the monomers. The SAXS profiles aredisplaced along the logarithmic axis for the sake of clarity. Thehomology model of the M. tuberculosis GlgE dimer(B) based on the S. coelicolor GlgE structure before(yellow) and after (red) rigid body refinement gave a root-mean-squaredeviation of 6.8 Å. The overall orientation of the dimer is similarto that in Figure 1.

Mentions: Although we are developing a good understandingof the structure of the S. coelicolor enzyme, a high-resolutionstructure of M. tuberculosis GlgE has proven to beelusive. It was already known that both enzymes formed dimers in solutionaccording to analytical ultracentrifugation.1,10 Toexplore their structural similarities further, they were both subjectedto SAXS analysis at several protein concentrations (Figure 4A and Figure S7 and Table S1 of the Supporting Information). The proteins had similarradii of gyration (Rg = 40 ± 1 Å)consistent with dimeric assemblies as expected. Further, the maximalsizes of the particles (Dmax) derivedfrom the p(r) function analysis(Figure S7 of the Supporting Information) were in good agreement with the maximal distance between surfaceamino acids of ∼130 Å observed in the crystallographichomodimer of S. coelicolor GlgE.


Structural insight into how Streptomyces coelicolor maltosyl transferase GlgE binds α-maltose 1-phosphate and forms a maltosyl-enzyme intermediate.

Syson K, Stevenson CE, Rashid AM, Saalbach G, Tang M, Tuukkanen A, Svergun DI, Withers SG, Lawson DM, Bornemann S - Biochemistry (2014)

SAXS analysis of GlgE proteins. The uppercurves (A) show the SAXSdata and fit for the S. coelicolor GlgE protein (χ= 1.03; experimental data colored blue and the theoretical profilecolored red on the basis of its X-ray crystal structure). The lowercurves show the M. tuberculosis GlgE SAXS data andfit of the theoretical profile of the initial homology model basedon the S. coelicolor GlgE crystal structure (χ= 1.34; experimental data colored green and fit colored blue) andafter the GENCRY rigid body refinement giving a significantly betterfit particularly in the range of 0.1–0.2 Å–1 (χ = 1.09; theoretical fit colored red), consistent with abetter relative orientation of the monomers. The SAXS profiles aredisplaced along the logarithmic axis for the sake of clarity. Thehomology model of the M. tuberculosis GlgE dimer(B) based on the S. coelicolor GlgE structure before(yellow) and after (red) rigid body refinement gave a root-mean-squaredeviation of 6.8 Å. The overall orientation of the dimer is similarto that in Figure 1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048318&req=5

fig4: SAXS analysis of GlgE proteins. The uppercurves (A) show the SAXSdata and fit for the S. coelicolor GlgE protein (χ= 1.03; experimental data colored blue and the theoretical profilecolored red on the basis of its X-ray crystal structure). The lowercurves show the M. tuberculosis GlgE SAXS data andfit of the theoretical profile of the initial homology model basedon the S. coelicolor GlgE crystal structure (χ= 1.34; experimental data colored green and fit colored blue) andafter the GENCRY rigid body refinement giving a significantly betterfit particularly in the range of 0.1–0.2 Å–1 (χ = 1.09; theoretical fit colored red), consistent with abetter relative orientation of the monomers. The SAXS profiles aredisplaced along the logarithmic axis for the sake of clarity. Thehomology model of the M. tuberculosis GlgE dimer(B) based on the S. coelicolor GlgE structure before(yellow) and after (red) rigid body refinement gave a root-mean-squaredeviation of 6.8 Å. The overall orientation of the dimer is similarto that in Figure 1.
Mentions: Although we are developing a good understandingof the structure of the S. coelicolor enzyme, a high-resolutionstructure of M. tuberculosis GlgE has proven to beelusive. It was already known that both enzymes formed dimers in solutionaccording to analytical ultracentrifugation.1,10 Toexplore their structural similarities further, they were both subjectedto SAXS analysis at several protein concentrations (Figure 4A and Figure S7 and Table S1 of the Supporting Information). The proteins had similarradii of gyration (Rg = 40 ± 1 Å)consistent with dimeric assemblies as expected. Further, the maximalsizes of the particles (Dmax) derivedfrom the p(r) function analysis(Figure S7 of the Supporting Information) were in good agreement with the maximal distance between surfaceamino acids of ∼130 Å observed in the crystallographichomodimer of S. coelicolor GlgE.

Bottom Line: The X-ray structures of α-maltose 1-phosphate bound to a D394A mutein and a β-2-deoxy-2-fluoromaltosyl-enzyme intermediate with a E423A mutein were determined.There are few examples of CAZy glycoside hydrolase family 13 members that have had their glycosyl-enzyme intermediate structures determined, and none before now have been obtained with a 2-deoxy-2-fluoro substrate analogue.The covalent modification of Asp394 was confirmed using mass spectrometry.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, John Innes Centre, Norwich Research Park , Norwich NR4 7UH, United Kingdom.

ABSTRACT
GlgE (EC 2.4.99.16) is an α-maltose 1-phosphate:(1→4)-α-d-glucan 4-α-d-maltosyltransferase of the CAZy glycoside hydrolase 13_3 family. It is the defining enzyme of a bacterial α-glucan biosynthetic pathway and is a genetically validated anti-tuberculosis target. It catalyzes the α-retaining transfer of maltosyl units from α-maltose 1-phosphate to maltooligosaccharides and is predicted to use a double-displacement mechanism. Evidence of this mechanism was obtained using a combination of site-directed mutagenesis of Streptomyces coelicolor GlgE isoform I, substrate analogues, protein crystallography, and mass spectrometry. The X-ray structures of α-maltose 1-phosphate bound to a D394A mutein and a β-2-deoxy-2-fluoromaltosyl-enzyme intermediate with a E423A mutein were determined. There are few examples of CAZy glycoside hydrolase family 13 members that have had their glycosyl-enzyme intermediate structures determined, and none before now have been obtained with a 2-deoxy-2-fluoro substrate analogue. The covalent modification of Asp394 was confirmed using mass spectrometry. A similar modification of wild-type GlgE proteins from S. coelicolor and Mycobacterium tuberculosis was also observed. Small-angle X-ray scattering of the M. tuberculosis enzyme revealed a homodimeric assembly similar to that of the S. coelicolor enzyme but with slightly differently oriented monomers. The deeper understanding of the structure-function relationships of S. coelicolor GlgE will aid the development of inhibitors of the M. tuberculosis enzyme.

Show MeSH
Related in: MedlinePlus