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Structural characterization of human aryl sulphotransferases.

ABSTRACT: Human aryl sulphotransferase (HAST) 1, HAST3, HAST4 and HAST4v share greater than 90% sequence identity, but vary markedly in their ability to catalyse the sulphonation of dopamine and p-nitrophenol. In order to investigate the amino acid(s) involved in determining differing substrate specificities of HASTs, a range of chimaeric HAST proteins were constructed. Analysis of chimaeric substrate specificities showed that enzyme affinities are mainly determined within the N-terminal end of each HAST protein, which includes two regions of high sequence divergence, termed Regions A (amino acids 44-107) and B (amino acids 132-164). To investigate the substrate-binding sites of HASTs further, site-directed mutagenesis was performed on HAST1 to change 13 individual residues within these two regions to the HAST3 equivalent. A single amino acid change in HAST1 (A146E) was able to change the specificity for p-nitrophenol to that of HAST3. The substrate specificity of HAST1 towards dopamine could not be converted into that of HAST3 with a single amino acid change. However, compared with wild-type HAST1, a number of the mutations resulted in interference with substrate binding, as shown by elevated Ki values towards the co-substrate 3'-phosphoadenosine 5'-phosphosulphate, and in some cases loss of activity towards dopamine. These findings suggest that a co-ordinated change of multiple amino acids in HAST proteins is needed to alter the substrate specificities of these enzymes towards dopamine, whereas a single amino acid at position 146 determines p-nitrophenol affinity. A HAST1 mutant was constructed to express a protein with four amino acids deleted (P87-P90). These amino acids were hypothesized to correspond to a loop region in close proximity to the substrate-binding pocket. Interestingly, the protein showed substrate specificities more similar to wild-type HAST3 than HAST1 and indicates an important role of these amino acids in substrate binding.

SUBMITTER: Brix LA

PROVIDER: S-EPMC1219970 | biostudies-other | 1999 Jan

REPOSITORIES: biostudies-other

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Project description:Npas4 is an activity dependent transcription factor which is responsible for gearing the expression of target genes involved in neuro-transmission. Despite the importance of Npas4 in many neuronal diseases, the tertiary structure of Npas4 protein along with its physico-chemical properties is limited. In the current study, first we perfomed the phylogenetic analysis of Npas4 and determined the content of hydrophobic, flexible and order-disorder promoting amino acids. The protein binding regions, post-translational modifications and crystallization propensity of Npas4 were predicted through different in-silico methods. The three dimensional model of Npas4 was predicted through LOMET, SPARSKS-X, I-Tasser, RaptorX, MUSTER and Pyhre and the best model was selected on the basis of Ramachandran plot, PROSA, and Qmean scores. The best model was then subjected to further refinement though MODREFINER. Finally the interacting partners of Npas4 were identified through STRING database. The phylogenetic analysis showed the human Npas4 gene to be closely related to other primates such as chimpanzees, monkey, gibbon. The physiochemical properties of Npas4 showed that it is an intrinsically disordered protein with N-terminal ordered region. The post-translational modification analyses indicated absence of acetylation and mannosylation sites. Three potential phosphorylation sites (S108, T130 and T136) were found in PAS A domain whilst a single phosphorylation site (S273) was present in PAS B domain. The predicted tertiary structure of Npas4 showed that bHLH domain and PAS domain possess tertiary structures while the rest of the protein exhibited disorder property. Protein-protein interaction analysis revealed NPas4 interaction with various proteins which are mainly involved in nuclear trafficking of proteins to cytoplasm, activity regulated gene transcription and neurodevelopmental disorders. Moreover the analysis also highlighted the direct relation to proteins involved in promoting neuronal survival, plasticity and cAMP responsive element binding protein proteins. The current study helps in understanding the physicochemical properties and reveals the neuro-modulatory role of Npas4 in crucial pathways involved in neuronal survival and neural signalling hemostasis.

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Structural characterization of human aryl sulphotransferases.

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