RESUMO
Sulfur, most abundantly found in the environment as sulfate (SO42-), is an essential element in metabolites required by all living cells, including amino acids, co-factors and vitamins. However, current understanding of the cellular delivery of SO42- at the molecular level is limited. CysZ has been described as a SO42- permease, but its sequence family is without known structural precedent. Based on crystallographic structure information, SO42- binding and flux experiments, we provide insight into the molecular mechanism of CysZ-mediated translocation of SO42- across membranes. CysZ structures from three different bacterial species display a hitherto unknown fold and have subunits organized with inverted transmembrane topology. CysZ from Pseudomonas denitrificans assembles as a trimer of antiparallel dimers and the CysZ structures from two other species recapitulate dimers from this assembly. Mutational studies highlight the functional relevance of conserved CysZ residues.
Assuntos
Alteromonadaceae/enzimologia , Alteromonadaceae/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Pseudomonas/enzimologia , Pseudomonas/metabolismo , Sulfatos/metabolismo , Biologia Computacional , Cristalografia por Raios X , Conformação Proteica , Multimerização ProteicaRESUMO
The CDP-alcohol phosphotransferase (CDP-AP) family of integral membrane enzymes catalyses the transfer of a substituted phosphate group from a CDP-linked donor to an alcohol acceptor. This is an essential reaction for phospholipid biosynthesis across all kingdoms of life, and it is catalysed solely by CDP-APs. Here we report the 2.0 Å resolution crystal structure of a representative CDP-AP from Archaeoglobus fulgidus. The enzyme (AF2299) is a homodimer, with each protomer consisting of six transmembrane helices and an N-terminal cytosolic domain. A polar cavity within the membrane accommodates the active site, lined with the residues from an absolutely conserved CDP-AP signature motif (D(1)xxD(2)G(1)xxAR...G(2)xxxD(3)xxxD(4)). Structures in the apo, CMP-bound, CDP-bound and CDP-glycerol-bound states define functional roles for each of these eight conserved residues and allow us to propose a sequential, base-catalysed mechanism universal for CDP-APs, in which the fourth aspartate (D4) acts as the catalytic base.
