Crystal structure of the enzyme CapF of Staphylococcus aureus reveals a unique architecture composed of two functional domains

2012 ◽  
Vol 443 (3) ◽  
pp. 671-680 ◽  
Author(s):  
Takamitsu Miyafusa ◽  
Jose M. M. Caaveiro ◽  
Yoshikazu Tanaka ◽  
Kouhei Tsumoto
Keyword(s):  
Staphylococcus Aureus ◽  
Capsular Polysaccharide ◽  
Binding Pocket ◽  
Therapeutic Agents ◽  
Titration Calorimetry ◽  
Enzymatic Reactions ◽  
Intermediate Species ◽  
Terminal Domain ◽  
Catalytic Loop ◽  
Important Virulence Factor

CP (capsular polysaccharide) is an important virulence factor during infections by the bacterium Staphylococcus aureus. The enzyme CapF is an attractive therapeutic candidate belonging to the biosynthetic route of CP of pathogenic strains of S. aureus. In the present study, we report two independent crystal structures of CapF in an open form of the apoenzyme. CapF is a homodimer displaying a characteristic dumb-bell-shaped architecture composed of two domains. The N-terminal domain (residues 1–252) adopts a Rossmann fold belonging to the short-chain dehydrogenase/reductase family of proteins. The C-terminal domain (residues 252–369) displays a standard cupin fold with a Zn2+ ion bound deep in the binding pocket of the β-barrel. Functional and thermodynamic analyses indicated that each domain catalyses separate enzymatic reactions. The cupin domain is necessary for the C3-epimerization of UDP-4-hexulose. Meanwhile, the N-terminal domain catalyses the NADPH-dependent reduction of the intermediate species generated by the cupin domain. Analysis by ITC (isothermal titration calorimetry) revealed a fascinating thermodynamic switch governing the attachment and release of the coenzyme NADPH during each catalytic cycle. These observations suggested that the binding of coenzyme to CapF facilitates a disorder-to-order transition in the catalytic loop of the reductase (N-terminal) domain. We anticipate that the present study will improve the general understanding of the synthesis of CP in S. aureus and will aid in the design of new therapeutic agents against this pathogenic bacterium.

scholarly journals Structure, dynamics, and inhibition of Staphylococcus aureus m1A22-tRNA methyltransferase

2021 ◽  
Author(s):  
Pamela Sweeney ◽  
Ashleigh Crowe ◽  
Abhishek Kumar ◽  
Dinesh Raju ◽  
Naveen B. Krishna ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Binding Pocket ◽  
Titration Calorimetry ◽  
Antibiotic Development ◽  
Functional Characterisation ◽  
Promising Target ◽  
Activity Measurements ◽  
Trna Methyltransferase ◽  
Dynamics Simulations ◽  
Rna Hairpin

The enzyme m1A22-tRNA methyltransferase (TrmK) catalyses the transfer of a methyl group from SAM to the N1 of adenine 22 in tRNAs. TrmK is essential for Staphylococcus aureus survival during infection, but has no homologue in mammals, making it a promising target for antibiotic development. Here we describe the structural and functional characterisation of S. aureus TrmK. Crystal structures are reported for S. aureus TrmK apoenzyme and in complexes with SAM and SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favourable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated by a large favourable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. Activity assays for S. aureus TrmK-catalysed methylation of WT and position 22 mutants of tRNALeu demonstrate that the enzyme requires an adenine at position 22 of the tRNA. Intriguingly, a small RNA hairpin of 18 nucleotides is methylated by TrmK depending on the position of the adenine. In-silico screening of compounds suggested plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92, in the vicinity of the SAM-binding site, as the sole residue modified. These results these results identify a cryptic binding pocket of S. aureus TrmK and lay the foundation for future structure-based drug discovery.

scholarly journals A Concerted Action of UBA5 C-Terminal Unstructured Regions Is Important for Transfer of Activated UFM1 to UFC1

2021 ◽  
Vol 22 (14) ◽  
pp. 7390
Author(s):  
Nicole Wesch ◽  
Frank Löhr ◽  
Natalia Rogova ◽  
Volker Dötsch ◽  
Vladimir V. Rogov
Keyword(s):  
Cellular Localization ◽  
Molecular Mechanisms ◽  
Biochemical Characterization ◽  
Effective Interaction ◽  
Regulatory Region ◽  
Titration Calorimetry ◽  
Enzymatic Reactions ◽  
Cellular Processes ◽  
Mechanism Of Interaction

Ubiquitin fold modifier 1 (UFM1) is a member of the ubiquitin-like protein family. UFM1 undergoes a cascade of enzymatic reactions including activation by UBA5 (E1), transfer to UFC1 (E2) and selective conjugation to a number of target proteins via UFL1 (E3) enzymes. Despite the importance of ufmylation in a variety of cellular processes and its role in the pathogenicity of many human diseases, the molecular mechanisms of the ufmylation cascade remains unclear. In this study we focused on the biophysical and biochemical characterization of the interaction between UBA5 and UFC1. We explored the hypothesis that the unstructured C-terminal region of UBA5 serves as a regulatory region, controlling cellular localization of the elements of the ufmylation cascade and effective interaction between them. We found that the last 20 residues in UBA5 are pivotal for binding to UFC1 and can accelerate the transfer of UFM1 to UFC1. We solved the structure of a complex of UFC1 and a peptide spanning the last 20 residues of UBA5 by NMR spectroscopy. This structure in combination with additional NMR titration and isothermal titration calorimetry experiments revealed the mechanism of interaction and confirmed the importance of the C-terminal unstructured region in UBA5 for the ufmylation cascade.

Inhibition of microbial adherence by sphinganine

1992 ◽  
Vol 38 (9) ◽  
pp. 983-985 ◽  
Author(s):  
Debra Jan Bibel ◽  
Raza Aly ◽  
Henry R. Shinefield
Keyword(s):  
Staphylococcus Aureus ◽  
Infectious Diseases ◽  
Therapeutic Agents ◽  
Eukaryotic Cells ◽  
Buccal Cells ◽  
Streptococcus Mitis ◽  
Buccal Epithelial Cells ◽  
Mucosal Cells ◽  
Microbial Adherence ◽  
Complex Sphingolipids

Sphingosines (precursors and degeneration products of complex sphingolipids) are mediators in membrane second-messenger cascades and in a wide variety of functions in eukaryotic cells. Sphingosines are also lethal for gram-positive microorganisms. In addition to its direct effect, sphinganine is here reported to affect the adherence of Streptococcus mitis to buccal epithelial cells and of Staphylococcus aureus to nasal mucosal cells after incubation for 90 min at 37 °C. When the bacteria were pretreated with 8.1, 16.2, 32.5, or (for Strep. mitis) 65 μM sphinganine for 60 min at 37 °C, adherence counts were reduced for Staph. aureus by 27, 37, and 60% and for Strep. mitis by 19, 44, 54, and 73%, respectively (p < 0.001). In contrast, pretreatment of buccal cells with 81.2 μM lipid increased adherence by 14% (p < 0.01), but no change occurred at either 16.2 or 325 μM lipid. These results further demonstrate the double-edged ability of sphingosines to regulate cellular activities and their potential as multifunctional therapeutic agents for infectious diseases. Key words: adherence, sphingosine, Staphylococcus aureus, Streptococcus mitis.

scholarly journals Determinants of the Endosomal Localization of Sorting Nexin 1

2005 ◽  
Vol 16 (4) ◽  
pp. 2049-2057 ◽  
Author(s):  
Qi Zhong ◽  
Martin J. Watson ◽  
Cheri S. Lazar ◽  
Andrea M. Hounslow ◽  
Jonathan P. Waltho ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Early Endosome ◽  
Nmr Structure ◽  
Sorting Nexin ◽  
High Affinity ◽  
Terminal Domain ◽  
Px Domain ◽  
Phox Homology ◽  
Phosphatidylinositol 3

The sorting nexin (SNX) family of proteins is characterized by sequence-related phox homology (PX) domains. A minority of PX domains bind with high affinity to phosphatidylinositol 3-phosphate [PI(3)P], whereas the majority of PX domains exhibit low affinity that is insufficient to target them to vesicles. SNX1 is located on endosomes, but its low affinity PX domain fails to localize in vivo. The NMR structure of the PX domain of SNX1 reveals an overall fold that is similar to high-affinity PX domains. However, the phosphatidylinositol (PI) binding pocket of the SNX1 PX domain is incomplete; regions of the pocket that are well defined in high-affinity PX domains are highly mobile in SNX1. Some of this mobility is lost upon binding PI(3)P. The C-terminal domain of SNX1 is a long helical dimer that localizes to vesicles but not to the early endosome antigen-1–containing vesicles where endogenous SNX1 resides. Thus, the obligate dimerization of SNX1 that is driven by the C-terminal domain creates a high-affinity PI binding species that properly targets the holo protein to endosomes.

scholarly journals Staphylococcus aureus CcpA Affects Virulence Determinant Production and Antibiotic Resistance

2006 ◽  
Vol 50 (4) ◽  
pp. 1183-1194 ◽  
Author(s):  
Kati Seidl ◽  
Martin Stucki ◽  
Martin Ruegg ◽  
Christiane Goerke ◽  
Christiane Wolz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Capsular Polysaccharide ◽  
Protein A ◽  
Virulence Gene ◽  
Exponential Growth Phase ◽  
Virulence Determinants ◽  
Resistance Level ◽  
Virulence Gene Expression ◽  
Oxacillin Resistance

ABSTRACT Carbon catabolite protein A (CcpA) is known to function as a major regulator of gene expression in different gram-positive organisms. Deletion of the ccpA homologue (saCOL1786) in Staphylococcus aureus was found to affect growth, glucose metabolization, and transcription of selected virulence determinants. In liquid culture, deletion of CcpA decreased the growth rate and yield; however, the effect was only transient during the exponential-growth phase as long as glucose was present in the medium. Depletion of glucose and production of lactate was delayed, while the level of excretion of acetate was less affected and was even higher in the mutant culture. On solid medium, in contrast, growth of the ΔccpA mutant resulted in smaller colonies containing a lower number of CFU per colony. Deletion of CcpA had an effect on the expression of important virulence factors of S. aureus by down-regulating RNAIII, the effector molecule of the agr locus, and altering the transcription patterns of hla, encoding α-hemolysin, and spa, encoding protein A. CcpA inactivation markedly reduced the oxacillin resistance levels in the highly methicillin-resistant S. aureus strain COLn and the teicoplanin resistance level in a glycopeptide-intermediate-resistant S. aureus strain. The presence of CcpA in the capsular polysaccharide serotype 5 (CP5)-producing strain Newman abolished capsule formation and decreased cap operon transcription in the presence of glucose. The staphylococcal CcpA thus not only is involved in the regulation of carbon metabolism but seems to function as a modulator of virulence gene expression as well.

Genotypic and phenotypic detection of capsular polysaccharide and biofilm formation in Staphylococcus aureus isolated from bovine milk collected from Brazilian dairy farms

2016 ◽  
Vol 40 (3-4) ◽  
pp. 97-106 ◽  
Author(s):  
Alessandra P.S. Salimena ◽  
Carla C. Lange ◽  
Cecilia Camussone ◽  
Marcelo Signorini ◽  
Luis F. Calvinho ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biofilm Formation ◽  
Capsular Polysaccharide ◽  
Bovine Milk ◽  
Dairy Farms

scholarly journals Identification and Characterization of thecps Locus of Streptococcus suis Serotype 2: the Capsule Protects against Phagocytosis and Is an Important Virulence Factor

1999 ◽  
Vol 67 (4) ◽  
pp. 1750-1756 ◽  
Author(s):  
Hilde E. Smith ◽  
Marloes Damman ◽  
Joeke van der Velde ◽  
Frans Wagenaar ◽  
Henk J. Wisselink ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Capsular Polysaccharide ◽  
Streptococcus Suis ◽  
Open Reading Frames ◽  
Transcriptional Unit ◽  
Polysaccharide Biosynthesis ◽  
Capsule Production ◽  
Important Virulence Factor ◽  
Isogenic Mutants

ABSTRACT To study the role of the capsule of Streptococcus suisserotype 2 in virulence, we generated two isogenic mutants disturbed in capsule production. For that purpose, we first cloned and characterized a major part of the capsular polysaccharide biosynthesis (cps) locus of S. suis serotype 2. Based on the established sequence, 14 open reading frames (ORFs), designated Orf2Z, Orf2Y, Orf2X, and Cps2A to Cps2K, were identified. Twelve ORFs belonged to a single transcriptional unit. The gene products of 11 of these ORFs showed similarity to proteins involved in polysaccharide biosynthesis of other gram-positive microorganisms. Nonencapsulated isogenic mutants were generated in the cps2B and cps2EF genes by insertional mutagenesis. In contrast to the wild-type S. suis serotype 2 strain, the nonencapsulated strains were highly sensitive to ingestion by porcine alveolar lung macrophages in vitro. More importantly, the nonencapsulated mutant strains were completely avirulent in young germfree pigs after intranasal inoculation. These observations indicate that the capsule of S. suis serotype 2 plays an essential role in the pathogenesis of S. suisserotype 2 infections.

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