Defluorination Capability of l-2-Haloacid Dehalogenases in the HAD-Like Hydrolase Superfamily Correlates with Active Site Compactness.

l-2-Haloacid dehalogenases, industrially and environmentally important enzymes that catalyse cleavage of the carbon-halogen bond in S-2-halocarboxylic acids, were known to hydrolyse chlorinated, brominated and iodinated substrates but no activity towards fluorinated compounds had been reported. A screen for novel dehalogenase activities revealed four l-2-haloacid dehalogenases capable of defluorination. We now report crystal structures for two of these enzymes, Bpro0530 and Rha0230, as well as for the related proteins PA0810 and RSc1362, which hydrolyse chloroacetate but not fluoroacetate, all at ∼2.2 Šresolution. Overall structure and active sites of these enzymes are highly similar. In molecular dynamics (MD) calculations, only the defluorinating enzymes sample more compact conformations, which in turn allow more effective interactions with the small fluorine atom. Structural constraints, based on X-ray structures and MD calculations, correctly predict the defluorination activity of the homologous enzyme ST2570.

Lysosomal integral membrane protein-2 (LIMP-2/SCARB2) is involved in lysosomal cholesterol export.

The intracellular transport of cholesterol is subject to tight regulation. The structure of the lysosomal integral membrane protein type 2 (LIMP-2, also known as SCARB2) reveals a large cavity that traverses the molecule and resembles the cavity in SR-B1 that mediates lipid transfer. The detection of cholesterol within the LIMP-2 structure and the formation of cholesterol-like inclusions in LIMP-2 knockout mice suggested the possibility that LIMP2 transports cholesterol in lysosomes. We present results of molecular modeling, crosslinking studies, microscale thermophoresis and cell-based assays that support a role of LIMP-2 in cholesterol transport. We show that the cavity in the luminal domain of LIMP-2 can bind and deliver exogenous cholesterol to the lysosomal membrane and later to lipid droplets. Depletion of LIMP-2 alters SREBP-2-mediated cholesterol regulation, as well as LDL-receptor levels. Our data indicate that LIMP-2 operates in parallel with Niemann Pick (NPC)-proteins, mediating a slower mode of lysosomal cholesterol export.