Ecto-5’-nucleotidase: Structure function relationships

Ecto-5’-nucleotidase (ecto-5’-NT) is attached via a GPI anchor to the extracellular membrane, where it hydrolyses AMP to adenosine and phosphate. Related 5’-nucleotidases exist in bacteria, where they are exported into the periplasmic space. X-ray structures of the 5’-nucleotidase from E. coli showed that the enzyme consists of two domains. The N-terminal domain coordinates two catalytic divalent metal ions, whereas the C-terminal domain provides the substrate specificity pocket for the nucleotides. Thus, the substrate binds at the interface of the two domains. Here, the currently available structural information on ecto-5’NT is reviewed in relation to the catalytic properties and enzyme function

A large hinge bending domain rotation is necessary for the catalytic function of Escherichia coli 5 `-nucleotidase

Two variants of Escherichia coli 5`-nucleotidase with disulfide bridges that were engineered to link the two domains of the protein were used to demonstrate that a large domain rotation is required for the catalytic mechanism of the enzyme. Kinetic analysis demonstrates that the variant trapped in the open form is almost inactive but can be activated up to 250-fold by reduction of the disulfide bridge. The second variant can adopt a closed but also a half-open conformation despite the presence of the cystine linkage. As a result of this flexibility, the mutant is still active in its oxidized state, although it shows a more pronounced substrate inhibition than the wild-type protein. A theoretical model is proposed that allows estimation of the flexibility of the proteins in the presence of the disulfide domain cross-link. Despite the unexpected residual flexibility of the trapped mutants, the enzymes could be used as conformational reporters in CD spectroscopy, revealing that the wild-type protein exists predominantly in an open conformation in solution. The kinetic, spectroscopic, and theoretical data are brought together to discuss the domain rotation in terms of the kinetic functioning of E. coli 5`-nucleotidase

Crystallization and preliminary characterization of three different crystal forms of human saposin C heterologously expressed in Pichia pastoris

Three different crystal forms were obtained of human saposin C. The structures could not be determined by molecular replacement using known solution structures of the protein as search models, supporting the notion of a highly flexible protein

Crystal structures of human saposins C and d implications for lipid recognition and membrane interactions

Human saposins are essential proteins required for degradation of sphingolipids and lipid antigen presentation. Despite the conserved structural organization of saposins, their distinct modes of interaction with biological membranes are not fully understood. We describe two crystal structures of human saposin C in an open configuration with unusual domain swapped homodimers. This form of SapC dimer supports the clip on model for SapC induced vesicle fusion. In addition, we present the crystal structure of SapD in two crystal forms. They reveal the monomer monomer interface for the SapD dimer, which was confirmed in solution by analytical ultracentrifugation. The crystal structure of SapD suggests that side chains of Lys10 and Arg17 are involved in initial association with the preferred anionic biological membranes by forming salt bridges with sulfate or phosphate lipid headgroup

Crystal structures of human saposins C and D: Implications for lipid recognition and membrane interactions

Human saposins are essential proteins required for degradation of sphingolipids and lipid antigen presentation. Despite the conserved structural organization of saposins, their distinct modes of interaction with biological membranes are not fully understood. We describe two crystal structures of human saposin C in an ``open`` configuration with unusual domain swapped homodimers. This form of SapC dimer supports the ``clip-on`` model for SapC-induced vesicle fusion. In addition, we present the crystal structure of SapD in two crystal forms. They reveal the monomer-monomer interface for the SapD dimer, which was confirmed in solution by analytical ultracentrifugation. The crystal structure of SapD suggests that side chains of Lys10 and Arg17 are involved in initial association with the preferred anionic biological membranes by forming salt bridges with sulfate or phosphate lipid headgroups

RPGR transcription studies in mouse and human tissues reveal a retina-specific isoform that is disrupted in a patient with X-linked retinitis pigmentosa.

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The Catalytic Mechanisms of Binuclear Metallohydrolases

With the aim of critically assessing the current models for metal ion assisted hydrolytic reaction mechanisms, an updated review of the current understanding of metallohydrolase-catalyzed reactions is presented. Focus is on four systems, purple acid phosphatases (PAPs), Ser/Thr protein phosphatases (PPs), 3′-5′ exonucleases, and 5′-nucleotidases (5′-NTs), which have contributed to major advancement of the understanding of the catalytic mechanisms that operate in such enzymes