ABSTRACT
S-adenosyl-L-homocysteine hydrolase (SAH hydrolase or SAHH) is a highly conserved enzyme that catalyses the reversible hydrolysis of SAH to L-homocysteine (HCY) and adenosine (ADO). High-resolution crystal structures have been reported for bacterial and plant SAHHs, but not mammalian SAHHs. Here, we report the first high-resolution crystal structure of mammalian SAHH (mouse SAHH) in complex with a reaction product (ADO) and with two reaction intermediate analogues-3'-keto-aristeromycin (3KA) and noraristeromycin (NRN)-at resolutions of 1.55, 1.55, and 1.65 Å. Each of the three structures constitutes a structural snapshot of one of the last three steps of the five-step process of SAH hydrolysis by SAHH. In the NRN complex, a water molecule, which is an essential substrate for ADO formation, is structurally identified for the first time as the candidate donor in a Michael addition by SAHH to the 3'-keto-4',5'-didehydroadenosine reaction intermediate. The presence of the water molecule is consistent with the reaction mechanism proposed by Palmer &Abeles in 1979. These results provide insights into the reaction mechanism of the SAHH enzyme.
Subject(s)
Adenosylhomocysteinase/chemistry , Adenosylhomocysteinase/metabolism , Adenosine/analogs & derivatives , Adenosine/chemistry , Adenosine/metabolism , Amino Acid Sequence , Animals , Binding Sites , Biocatalysis , Crystallography, X-Ray , Hydrolysis , Mice , Molecular Dynamics Simulation , Molecular Sequence Data , Protein Structure, Tertiary , S-Adenosylhomocysteine/metabolism , Sequence Alignment , Substrate SpecificityABSTRACT
S-adenosyl-L-homocysteine hydrolase (SAHH; EC 3.3.1.1) catalyzes the reversible hydrolysis of S-adenosyl-L-homocysteine to adenosine and L-homocysteine. For crystallographic investigations, mouse SAHH (MmSAHH) was overexpressed in bacterial cells and crystallized using the hanging-drop vapour-diffusion method in the presence of the reaction product adenosine. X-ray diffraction data to 1.55 A resolution were collected from an orthorhombic crystal form belonging to space group I222 with unit-cell parameters a = 100.64, b = 104.44, c = 177.31 A. Structural analysis by molecular replacement is in progress.
Subject(s)
Adenosylhomocysteinase/chemistry , Adenosylhomocysteinase/genetics , Adenosylhomocysteinase/isolation & purification , Animals , Crystallography, X-Ray , Gene Expression , MiceABSTRACT
Coronin cDNA was cloned from the plasmodia of Physarum polycephalum. The amino acid sequence deduced from the cDNA was comprised of 449 residues and showed 60% identity to that of Dictyostelium discoideum coronin. Southern blot analysis suggested that the coronin gene present in the P. polycephalum genome might be a single copy. Coronin was expressed in diploid plasmodia, while it was not detected in haploid amoebae or spores.
Subject(s)
4-Butyrolactone/analogs & derivatives , DNA, Complementary/genetics , Gene Expression Regulation, Developmental , Life Cycle Stages/genetics , Physarum polycephalum/growth & development , Physarum polycephalum/genetics , 4-Butyrolactone/chemistry , 4-Butyrolactone/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , Molecular Sequence Data , Sequence AlignmentABSTRACT
We have evaluated the relationship between carotid atherosclerotic change and periventricular hyper intensity (PVH). PVH was studied in 66 cases with cerebral thrombosis, comparing them with another group of age-matched controls, which consisted of 29 cases with hypertension, diabetes, and hypercholesteremia. MRI (fluid attenuated inversion recovery) and B-mode carotid ultrasonography of each lesion were analyzed. Thrombosis lesions, compatible with neurological manifestation were divided into two types, cerebral cortical type (including centrum semiovale type) and small infarction in the deep subcortical type. PVH was classified into 4 grades, none, rims/caps, patchy and diffuse. Smooth PVH was, adjoining the anterior/posterior angles and the margins of the lateral ventricles, were defined as caps and rims. Irregular PVH areas, confluent with each other, were defined as patchy, while diffuse PVH areas extending below the cortex beyond the level of the corpus callosum were defined as diffuse. Carotid atherosclerosis was evaluated using B-mode carotid ultrasonography. The severity of carotid atherosclerosis was assessed by using two indicators; incidence of carotid atherosclerosis and maximum percentage diameter of the stenosis areas. Patchy and diffuse type PVH was frequent in the thrombosis group. On B-mode carotid ultrasonography, diffuse PVH was prominent in patients with stenotic change and high maximum percentage of stenosis, but none/rims/caps PVH was accompanied by variable B-mode carotid ultrasonographical findings. Six patients had ulcerated plaques and they suffered more frequently with diffuse PVH. Diffuse PVH was more frequent in cases with severe carotid stenosis than in other PVH types. These findings suggested that large vessel atherosclerosis could result in diffuse PVH.
