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1.  Crystal structure and functional insights into uracil-DNA glycosylase inhibition by phage ϕ29 DNA mimic protein p56 
Nucleic Acids Research  2013;41(13):6761-6773.
Uracil-DNA glycosylase (UDG) is a key repair enzyme responsible for removing uracil residues from DNA. Interestingly, UDG is the only enzyme known to be inhibited by two different DNA mimic proteins: p56 encoded by the Bacillus subtilis phage ϕ29 and the well-characterized protein Ugi encoded by the B. subtilis phage PBS1/PBS2. Atomic-resolution crystal structures of the B. subtilis UDG both free and in complex with p56, combined with site-directed mutagenesis analysis, allowed us to identify the key amino acid residues required for enzyme activity, DNA binding and complex formation. An important requirement for complex formation is the recognition carried out by p56 of the protruding Phe191 residue from B. subtilis UDG, whose side-chain is inserted into the DNA minor groove to replace the flipped-out uracil. A comparative analysis of both p56 and Ugi inhibitors enabled us to identify their common and distinctive features. Thereby, our results provide an insight into how two DNA mimic proteins with different structural and biochemical properties are able to specifically block the DNA-binding domain of the same enzyme.
PMCID: PMC3711442  PMID: 23671337
2.  NADP+ Binding to the Regulatory Subunit of Methionine Adenosyltransferase II Increases Intersubunit Binding Affinity in the Hetero-Trimer 
PLoS ONE  2012;7(11):e50329.
Mammalian methionine adenosyltransferase II (MAT II) is the only hetero-oligomer in this family of enzymes that synthesize S-adenosylmethionine using methionine and ATP as substrates. Binding of regulatory β subunits and catalytic α2 dimers is known to increase the affinity for methionine, although scarce additional information about this interaction is available. This work reports the use of recombinant α2 and β subunits to produce oligomers showing kinetic parameters comparable to MAT II purified from several tissues. According to isothermal titration calorimetry data and densitometric scanning of the stained hetero-oligomer bands on denatured gels, the composition of these oligomers is that of a hetero-trimer with α2 dimers associated to single β subunits. Additionally, the regulatory subunit is able to bind NADP+ with a 1∶1 stoichiometry, the cofactor enhancing β to α2-dimer binding affinity. Mutants lacking residues involved in NADP+ binding and N-terminal truncations of the β subunit were able to oligomerize with α2-dimers, although the kinetic properties appeared altered. These data together suggest a role for both parts of the sequence in the regulatory role exerted by the β subunit on catalysis. Moreover, preparation of a structural model for the hetero-oligomer, using the available crystal data, allowed prediction of the regions involved in β to α2-dimer interaction. Finally, the implications that the presence of different N-terminals in the β subunit could have on MAT II behavior are discussed in light of the recent identification of several splicing forms of this subunit in hepatoma cells.
PMCID: PMC3506619  PMID: 23189196
3.  Crystallization and preliminary X-ray diffraction analysis of the fructofuranosidase from Xanthophyllomyces dendrorhous  
The invertase from X. dendrorhous has been purified, deglycosylated and crystallized and diffraction data have been collected to 2.3 Å resolution.
Xanthophyllomyces dendrorhous invertase is an extracellular enzyme that releases β-fructose from the nonreducing termini of various β-d-fructofuranoside substrates. Its ability to produce neokestose by transglycosylation makes this enzyme an interesting research target for applications in industrial biotechnology. The native enzyme, which is highly glycosylated, failed to crystallize. Therefore, it was submitted to EndoH deglycosylating treatment and crystals were grown by vapour-diffusion methods. The crystals belonged to space group P21212, with unit-cell parameters a = 75.29, b = 204.93, c = 146.25 Å. Several diffraction data sets were collected using a synchrotron source. Self-rotation function and gel-filtration experiments suggested that the enzyme is a dimer with twofold symmetry.
PMCID: PMC3001643  PMID: 21045290
yeast invertase; β-fructofuranosidases; glycoside hydrolase family 32
4.  Fructo-Oligosaccharide Synthesis by Mutant Versions of Saccharomyces cerevisiae Invertase ▿† 
Applied and Environmental Microbiology  2011;77(17):6148-6157.
Efficient enzymatic synthesis of tailor-made prebiotic fructo-oligosaccharides (FOS) used in functional food formulation is a relevant biotechnological objective. We have engineered the Saccharomyces cerevisiae invertase (Suc2) to improve its transferase activity and to identify the enzymatic determinants for product specificity. Amino acid replacement (W19Y, N21S, N24S) within a conserved motif (β-fructosidase) specifically increased the synthesis of 6-kestose up to 10-fold. Mutants with lower substrate (sucrose) affinity produced FOS with longer half-lives. A mutation (P205V) adjacent to another conserved motif (EC) caused a 6-fold increment in 6-kestose yield. Docking studies with a Suc2 modeled structure defined a putative acceptor substrate binding subsite constituted by Trp 291 and Asn 228. Mutagenesis studies confirmed the implication of Asn 228 in directing the orientation of the sucrose molecule for the specific synthesis of β(2,6) linkages.
PMCID: PMC3165384  PMID: 21764973
5.  Crystallization and preliminary X-ray crystallographic analysis of β-galactosidase from Kluyveromyces lactis  
β-Galactosidase from K. lactis has been expressed in S. cerevisiae, purified by affinity chromatography and crystallized in its native form.
β-Galactosidase from Kluyveromyces lactis catalyses the hydrolysis of the β-­galactosidic linkage in lactose. Owing to its many industrial applications, the biotechnological potential of this enzyme is substantial. This protein has been expressed in yeast and purified for crystallization trials. However, optimization of the best crystallization conditions yielded crystals with poor diffraction quality that precluded further structural studies. Finally, the crystal quality was improved using the streak-seeding technique and a complete diffraction data set was collected at 2.8 Å resolution.
PMCID: PMC2833041  PMID: 20208165
yeast; β-galactosidases; glycoside hydrolase family 2
6.  Crystallization and preliminary X-ray diffraction analysis of inositol 1,3,4,5,6-pentakisphosphate kinase from Arabidopsis thaliana  
Inositol 1,3,4,5,6-pentakisphosphate kinase from A. thaliana has been expressed in E. coli, purified and crystallized and diffraction data have been collected to 2.3 Å resolution. Two heavy-atom crystal derivatives are under study.
Inositol 1,3,4,5,6-pentakisphosphate kinase (IP5 2-K) is an enzyme involved in inositol metabolism that synthesizes IP6 (inositol 1,2,3,4,5,6-hexakisphosphate) from inositol 1,3,4,5,6-pentakisphosphate (IP5) and ATP. IP6 is the major phosphorus reserve in plants, while in mammals it is involved in multiple cellular events such as DNA editing and chromatin remodelling. In addition, IP6 is the precursor of other highly phosphorylated inositols which also play highly relevant roles. IP5 2-K is the only enzyme that phosphorylates the 2-OH axial position of the inositide and understanding its molecular mechanism of substrate specificity is of great interest in cell biology. IP5 2-K from Arabidopsis thaliana has been expressed in Escherichia coli as two different fusion proteins and purified. Both protein preparations yielded crystals of different quality, always in the presence of IP6. The best crystals obtained for X-ray crystallographic analysis belonged to space group P212121, with unit-cell parameters a = 58.124, b = 113.591, c = 142.478 Å. Several diffraction data sets were collected for the native enzyme and two heavy-atom derivatives using a synchrotron source.
PMCID: PMC2805549  PMID: 20057083
inositol kinases; phytic acid; inositol 1,2,3,4,5,6-hexakisphosphate; inositol 1,3,4,5,6-pentakisphosphate kinase
7.  Crystallization and preliminary X-ray diffraction analysis of the fructofuranosidase from Schwanniomyces occidentalis  
The invertase from Schwanniomyces occidentalis has been expressed in Saccharomyces cerevisiae, purified and crystallized. The wild-type enzyme was also purified and crystallized and diffraction data were collected to 2.9 Å resolution.
Schwanniomyces occidentalis invertase is an extracellular enzyme that releases β-fructose from the nonreducing termini of various β-d-fructofuranoside substrates. Its ability to produce 6-kestose by transglycosylation makes this enzyme an interesting research target for applications in industrial biotechnology. The enzyme has been expressed in Saccharomyces cerevisiae. Recombinant and wild-type forms, which showed different glycosylation patterns, were crystallized by vapour-diffusion methods. Although crystallization trials were conducted on both forms of the protein, crystals suitable for X-ray crystallographic analyses were only obtained from the wild-type enzyme. The crystals belonged to space group P212121, with unit-cell parameters a = 105.78, b = 119.49, c = 137.68 Å. A diffraction data set was collected using a synchrotron source. Self-rotation function and sedimentation-velocity experiments suggested that the enzyme was dimeric with twofold symmetry.
PMCID: PMC2777049  PMID: 19923741
yeast invertases; fructofuranosidases; glycoside hydrolase family 32
8.  New Insights into the Fructosyltransferase Activity of Schwanniomyces occidentalis β-Fructofuranosidase, Emerging from Nonconventional Codon Usage and Directed Mutation▿  
Applied and Environmental Microbiology  2010;76(22):7491-7499.
Schwanniomyces occidentalis β-fructofuranosidase (Ffase) releases β-fructose from the nonreducing ends of β-fructans and synthesizes 6-kestose and 1-kestose, both considered prebiotic fructooligosaccharides. Analyzing the amino acid sequence of this protein revealed that it includes a serine instead of a leucine at position 196, caused by a nonuniversal decoding of the unique mRNA leucine codon CUG. Substitution of leucine for Ser196 dramatically lowers the apparent catalytic efficiency (kcat/Km) of the enzyme (approximately 1,000-fold), but surprisingly, its transferase activity is enhanced by almost 3-fold, as is the enzymes' specificity for 6-kestose synthesis. The influence of 6 Ffase residues on enzyme activity was analyzed on both the Leu196/Ser196 backgrounds (Trp47, Asn49, Asn52, Ser111, Lys181, and Pro232). Only N52S and P232V mutations improved the transferase activity of the wild-type enzyme (about 1.6-fold). Modeling the transfructosylation products into the active site, in combination with an analysis of the kinetics and transfructosylation reactions, defined a new region responsible for the transferase specificity of the enzyme.
PMCID: PMC2976189  PMID: 20851958

Results 1-8 (8)