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Carvalho, AL, Goyal A, Prates JAM, Bolam DN, Gilbert HJ, Pires VMR, Ferreira LMA, Planas A, Romao MJ, Fontes C.  2004.  The family 11 carbohydrate-binding module of Clostridium thermocellum Lic26A-Cel5E accommodates beta-1,4- and beta-1,3-1,4-mixed linked glucans at a single binding site. Journal of Biological Chemistry. 279:34785-34793., Number 33 AbstractWebsite
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Ribeiro, T, Santos-Silva T, Alves VD, Dias FMV, Luis AS, Prates JAM, Ferreira LMA, Romao MJ, Fontes CMGA.  2010.  Family 42 carbohydrate-binding modules display multiple arabinoxylan-binding interfaces presenting different ligand affinities. Biochimica Et Biophysica Acta-Proteins and Proteomics. 1804:2054-2062., Number 10 AbstractWebsite
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Santos-Silva, T, Trincao J, Carvalho AL, Bonifacio C, Auchere F, Raleiras P, Moura I, Moura JJG, Romao MJ.  2006.  The first crystal structure of class III superoxide reductase from Treponema pallidum. Journal of Biological Inorganic Chemistry. 11:548-558., Number 5 AbstractWebsite
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Leisico, F, V. Vieira D, Figueiredo TA, Silva M, Cabrita EJ, Sobral RG, Ludovice AM, Trincão J, Romão MJ, de Lencastre H, Santos-Silva T.  2018.  First insights of peptidoglycan amidation in Gram-positive bacteria - the high-resolution crystal structure of Staphylococcus aureus glutamine amidotransferase GatD, 2018. Scientific Reports. 8(1):5313. AbstractWebsite

Gram-positive bacteria homeostasis and antibiotic resistance mechanisms are dependent on the intricate architecture of the cell wall, where amidated peptidoglycan plays an important role. The amidation reaction is carried out by the bi-enzymatic complex MurT-GatD, for which biochemical and structural information is very scarce. In this work, we report the first crystal structure of the glutamine amidotransferase member of this complex, GatD from Staphylococcus aureus, at 1.85 Å resolution. A glutamine molecule is found close to the active site funnel, hydrogen-bonded to the conserved R128. In vitro functional studies using 1H-NMR spectroscopy showed that S. aureus MurT-GatD complex has glutaminase activity even in the absence of lipid II, the MurT substrate. In addition, we produced R128A, C94A and H189A mutants, which were totally inactive for glutamine deamidation, revealing their essential role in substrate sequestration and catalytic reaction. GatD from S. aureus and other pathogenic bacteria share high identity to enzymes involved in cobalamin biosynthesis, which can be grouped in a new sub-family of glutamine amidotransferases. Given the ubiquitous presence of GatD, these results provide significant insights into the molecular basis of the so far undisclosed amidation mechanism, contributing to the development of alternative therapeutics to fight infections.

Coelho, C, Mahro M, Trincao J, Carvalho ATP, Ramos MJ, Terao M, Garattini E, Leimkuehler S, Romao MJ.  2012.  The First Mammalian Aldehyde Oxidase Crystal Structure INSIGHTS INTO SUBSTRATE SPECIFICITY. Journal of Biological Chemistry. 287:40690-40702., Number 48 AbstractWebsite
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Raaijmakers, HCA, Romao MJ.  2006.  Formate-reduced E-coli formate dehydrogenase H: the reinterpretation of the crystal structure suggests a new reaction mechanism. Journal of Biological Inorganic Chemistry. 11:849-854., Number 7 AbstractWebsite
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Vidossich, P, Castañeda Moreno LE, Mota C, de Sanctis D, Miscione GP, De Vivo M.  2020.  Functional Implications of Second-Shell Basic Residues for dUTPase DR2231 Enzymatic Specificity, 2020. ACS CatalysisACS Catalysis. 10(23):13825-13833.: American Chemical Society AbstractWebsite

Nucleotide-processing enzymes are key players in biological processes. They often operate through high substrate specificity for catalysis. How such specificity is achieved is unclear. Here, we dealt with this question by investigating all-α dimeric deoxyuridine triphosphate nucleotidohydrolases (dUTPases). Typically, these dUTPases hydrolyze either dUTP or deoxyuridine diphosphate (dUDP) substrates. However, the dUTPase enzyme DR2231 from Deinococcus radiodurans selectively hydrolyzes dUTP only, and not dUDP. By means of extended classical molecular dynamics simulations and quantum chemical calculations, we show that DR2231 achieves this specificity for dUTP via second-shell basic residues that, together with the two catalytic magnesium ions, contribute to properly orienting the γ-phosphate of dUTP in a prereactive state. This allows a nucleophilic water to be correctly placed and activated in order to perform substrate hydrolysis. We show that this enzymatic mechanism is not viable when dUDP is bound to DR2231. Importantly, in several other dUTPases capable of hydrolyzing either dUTP or dUDP, we detected that active site second-shell basic residues are more in number, anchoring the β-phosphate of the nucleotide substrate too, in contrast to what is observed in DR2231. Thus, strategically located basic second-shell residues mediate precise reactant positioning at the catalytic site, determining substrate specificity in dUTPases and possibly in other structurally similar nucleotide-processing metalloenzymes.Nucleotide-processing enzymes are key players in biological processes. They often operate through high substrate specificity for catalysis. How such specificity is achieved is unclear. Here, we dealt with this question by investigating all-α dimeric deoxyuridine triphosphate nucleotidohydrolases (dUTPases). Typically, these dUTPases hydrolyze either dUTP or deoxyuridine diphosphate (dUDP) substrates. However, the dUTPase enzyme DR2231 from Deinococcus radiodurans selectively hydrolyzes dUTP only, and not dUDP. By means of extended classical molecular dynamics simulations and quantum chemical calculations, we show that DR2231 achieves this specificity for dUTP via second-shell basic residues that, together with the two catalytic magnesium ions, contribute to properly orienting the γ-phosphate of dUTP in a prereactive state. This allows a nucleophilic water to be correctly placed and activated in order to perform substrate hydrolysis. We show that this enzymatic mechanism is not viable when dUDP is bound to DR2231. Importantly, in several other dUTPases capable of hydrolyzing either dUTP or dUDP, we detected that active site second-shell basic residues are more in number, anchoring the β-phosphate of the nucleotide substrate too, in contrast to what is observed in DR2231. Thus, strategically located basic second-shell residues mediate precise reactant positioning at the catalytic site, determining substrate specificity in dUTPases and possibly in other structurally similar nucleotide-processing metalloenzymes.