scholarly journals Structural Basis for Peptide Substrate Specificities of Glycosyltransferase GalNAc-T2

ACS Catalysis ◽  
2021 ◽  
pp. 2977-2991
Author(s):  
Sai Pooja Mahajan ◽  
Yashes Srinivasan ◽  
Jason W. Labonte ◽  
Matthew P. DeLisa ◽  
Jeffrey J. Gray
2007 ◽  
Vol 190 (3) ◽  
pp. 1108-1117 ◽  
Author(s):  
Taisuke Wakamatsu ◽  
Noriko Nakagawa ◽  
Seiki Kuramitsu ◽  
Ryoji Masui

ABSTRACT ADP-ribose (ADPR) is one of the main substrates of Nudix proteins. Among the eight Nudix proteins of Thermus thermophilus HB8, we previously determined the crystal structure of Ndx4, an ADPR pyrophosphatase (ADPRase). In this study we show that Ndx2 of T. thermophilus also preferentially hydrolyzes ADPR and flavin adenine dinucleotide and have determined its crystal structure. We have determined the structures of Ndx2 alone and in complex with Mg2+, with Mg2+ and AMP, and with Mg2+ and a nonhydrolyzable ADPR analogue. Although Ndx2 recognizes the AMP moiety in a manner similar to those for other ADPRases, it recognizes the terminal ribose in a distinct manner. The residues responsible for the recognition of the substrate in Ndx2 are not conserved among ADPRases. This may reflect the diversity in substrate specificity among ADPRases. Based on these results, we propose the classification of ADPRases into two types: ADPRase-I enzymes, which exhibit high specificity for ADPR; and ADPRase-II enzymes, which exhibit low specificity for ADPR. In the active site of the ternary complexes, three Mg2+ ions are coordinated to the side chains of conserved glutamate residues and water molecules. Substitution of Glu90 and Glu94 with glutamine suggests that these residues are essential for catalysis. These results suggest that ADPRase-I and ADPRase-II enzymes have nearly identical catalytic mechanisms but different mechanisms of substrate recognition.


Biochemistry ◽  
1987 ◽  
Vol 26 (14) ◽  
pp. 4461-4466 ◽  
Author(s):  
Nancy E. Thomas ◽  
H. Neal Bramson ◽  
W. Todd Miller ◽  
E. T. Kaiser

2021 ◽  
Author(s):  
Yuchen Zhang ◽  
Keisuke Hamada ◽  
Dinh Thanh Nguyen ◽  
Sumika Inoue ◽  
Masayuki Satake ◽  
...  

Prenylation plays an important role in diversifying structure and function of secondary metabolites. Although several cyanobactin prenyltransferases have been characterized, their modes of action are mainly limited to the modification of electron-rich hetero atoms. Here we report a unique prenyltransferase originating from Limnothrix sp. CACIAM 69d, referred to as LimF, which catalyzes an unprecedented His-C-geranylation. Interestingly, LimF executes the geranylation on not only its native peptide substrate but also a wide range of exotic peptides, including thioether-closed macrocycles. We have also serendipitously uncovered an ability of Tyr-O-geranylation as the secondary function of LimF, indicating it is an unusual bifunctional prenyltransferase. Crystallographic analysis of LimF complexed with a pentapeptide substrate and a prenyl donor analog provides structural basis for its unique His recognition and its bifunctionality. Lastly, we show the LimF’s prenylation ability on various bioactive molecules containing an imidazole group, highlighting its potential as a versatile biocatalyst for site-specific geranylation.


eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Justin J Miller ◽  
Ishaan T Shah ◽  
Jayda Hatten ◽  
Yasaman Barekatain ◽  
Elizabeth A Mueller ◽  
...  

Carboxy ester prodrugs are widely employed to increase oral absorption and potency of phosphonate antibiotics. Prodrugging can mask problematic chemical features that prevent cellular uptake and may enable tissue specific compound delivery. However, many carboxy ester promoieties are rapidly hydrolyzed by serum esterases, limiting their therapeutic potential. While carboxy ester-based prodrug targeting is feasible, it has seen limited use in microbes as microbial esterase specific promoieties have not been described. Here we identify the bacterial esterases, GloB and FrmB, that activate carboxy ester prodrugs in Staphylococcus aureus. Additionally, we determine the substrate specificities for FrmB and GloB and demonstrate the structural basis of these preferences. Finally, we establish the carboxy ester substrate specificities of human and mouse sera, ultimately identifying several promoieties likely to be serum esterase-resistant and microbially labile. These studies will enable structure-guided design of anti-staphylococcal promoieties and expand the range of molecules to target staphylococcal pathogens.


2016 ◽  
Vol 44 (1) ◽  
pp. 61-67 ◽  
Author(s):  
Ramon Hurtado-Guerrero

Protein O-GalNAcylation is an abundant post-translational modification and predicted to occur in over 80% of the proteins passing through the Golgi apparatus. This modification is driven by 20 polypeptide GaINAc (N-acetylgalactosamine)-transferases (GalNAc-Ts), which are unique in that they possess both catalytic and lectin domains. The peptide substrate specificities of GalNAc-Ts are still poorly defined and our understanding of the sequence and structural features that direct O-glycosylation of proteins is limited. Part of this may be attributed to the complex regulation by coordinated action of multiple GalNAc-T isoforms, and part of this may also be attributed to the two functional domains of GalNAc-Ts that both seems to be involved in directing the substrate specificities. Recent studies have resulted in 3D structures of GalNAc-Ts and determination of the reaction mechanism of this family of enzymes. Key advances include the trapping of binary/ternary complexes in combination with computational simulations and AFM/small-SAXS experiments, which have allowed for the dissection of the reaction coordinates and the mechanism by which the lectin domains modulate the glycosylation. These studies not only broaden our knowledge of the modes-of-action of this family of enzymes but also open up potential avenues for the rational design of effective and selective inhibitors of O-glycosylation.


2015 ◽  
Vol 197 (22) ◽  
pp. 3583-3591 ◽  
Author(s):  
John R. Brannon ◽  
Jenny-Lee Thomassin ◽  
Samantha Gruenheid ◽  
Hervé Le Moual

ABSTRACTBacterial proteases contribute to virulence by cleaving host or bacterial proteins to promote survival and dissemination. Omptins are a family of proteases embedded in the outer membrane of Gram-negative bacteria that cleave various substrates, including host antimicrobial peptides, with a preference for cleaving at dibasic motifs. OmpT, the enterohemorrhagicEscherichia coli(EHEC) omptin, cleaves and inactivates the human cathelicidin LL-37. Similarly, the omptin CroP, found in the murine pathogenCitrobacter rodentium, which is used as a surrogate model to study human-restricted EHEC, cleaves the murine cathelicidin-related antimicrobial peptide (CRAMP). Here, we compared the abilities of OmpT and CroP to cleave LL-37 and CRAMP. EHEC OmpT degraded LL-37 and CRAMP at similar rates. In contrast,C. rodentiumCroP cleaved CRAMP more rapidly than LL-37. The different cleavage rates of LL-37 and CRAMP were independent of the bacterial background and substrate sequence specificity, as OmpT and CroP have the same preference for cleaving at dibasic sites. Importantly, LL-37 was α-helical and CRAMP was unstructured under our experimental conditions. By altering the α-helicity of LL-37 and CRAMP, we found that decreasing LL-37 α-helicity increased its rate of cleavage by CroP. Conversely, increasing CRAMP α-helicity decreased its cleavage rate. This structural basis for CroP substrate specificity highlights differences between the closely related omptins ofC. rodentiumandE. coli. In agreement with previous studies, this difference in CroP and OmpT substrate specificity suggests that omptins evolved in response to the substrates present in their host microenvironments.IMPORTANCEOmptins are recognized as key virulence factors for various Gram-negative pathogens. Their localization to the outer membrane, their active site facing the extracellular environment, and their unique catalytic mechanism make them attractive targets for novel therapeutic strategies. Gaining insights into similarities and variations between the different omptin active sites and subsequent substrate specificities will be critical to develop inhibitors that can target multiple omptins. Here, we describe subtle differences between the substrate specificities of two closely related omptins, CroP and OmpT. This is the first reported example of substrate conformation acting as a structural determinant for omptin activity between OmpT-like proteases.


2020 ◽  
Author(s):  
Justin Miller ◽  
Ishaan T Shah ◽  
Jayda Hatten ◽  
Yasaman Barekatain ◽  
Elizabeth A Mueller ◽  
...  

Carboxy ester prodrugs have been widely employed as a means to increase oral absorption and potency of phosphonate antibiotics. Prodrugging can successfully mask problematic chemical features that prevent cellular uptake and can be used to target delivery of compounds to specific tissues. However, many carboxy ester promoieties are rapidly hydrolyzed by serum esterases, curbing their potential therapeutic applications. While carboxy ester-based prodrug targeting is feasible, it has been limited in microbes due to a paucity of information about the selectivity of microbial esterases. Here we identify the bacterial esterases, GloB and FrmB, that are required for carboxy ester prodrug activation in Staphylococcus aureus. Additionally, we determine the substrate specificities for FrmB and GloB and demonstrate the structural basis of these preferences. Finally, we establish the carboxy ester substrate specificities of human and mouse sera, identifying several promoieties likely to be serum esterase-resistant while still being microbially labile. These studies lay the groundwork for structure guided design of antistaphyloccal promoieties, enabling a massive expansion of the antistaphyloccal druggable space.


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