The critical role of His48 in mouse cytosolic sulfotransferase SULT2A8 for the 7α-hydroxyl sulfation of bile acids

Abstract

Members of the cytosolic sulfotransferase (SULT) SULT2A subfamily are known to be critically involved in the homeostasis of steroids and bile acids. SULT2A8, a 7α-hydroxyl bile acid-preferring mouse SULT, has been identified as the major enzyme responsible for the mouse-specific 7-O-sulfation of bile acids. Interestingly, SULT2A8 lacks a conservative catalytic His residue at position 99th. The catalytic mechanism underlying the SULT2A8-mediated 7-O-sulfation of bile acids thus remained unclear. In this study, we performed a mutational analysis in order to gain insight into this yet-unresolved issue. Results obtained revealed two amino acid residues, His48 and Leu99, that are unique to the mouse SULT2A8, but not other SULTs, are essential for its 7-O-sulfating activity toward bile acids. These findings suggested that substitutions of two amino acids, which might have occurred during the evolution of the mouse SULT2A8 gene, endowed mouse SULT2A8 the capacity to catalyze the 7-O-sulfation of bile acids.

Structure of mouse cytosolic sulfotransferase SULT2A8 provides insight into sulfonation of 7α-hydroxyl bile acids

“…The overall effects of these two substitution mutants are comparable with the study of Shimohira et al. ( 50 ), although the extent of activity reduction is less dramatic in our study. The discrepancy could likely be due to the differences in assay buffer condition.…”

“…S4). Our model provides structural evidence for how His48 acts as a catalytic residue for the deprotonation of the substrate as proposed by the previous study (50). Results from our sitedirected mutagenesis studies further imply that both Lys44 and His48 are key residues for 7α-OH sulfonation, although only 40% enzyme activity was retained when either residue was mutated.…”

“…The importance of His48 in mSULT2A8 activity has previously been studied, that is, substitutions of His48 with asparagine and threonine led to 10% residual enzyme activity and nondetectable activity, respectively ( 50 ). Here, we carried out a thorough mutagenesis analysis to validate the functional roles of the residues in the active site and on loop 3 as revealed from the crystal structure of SULT2A8.…”

“…It also explains why crystallization of SULT2A8 in the absence of either cofactor or substrate could not be obtained. In mouse, the hepatic levels of PAPS (∼20 μM) ( 78 ) and T-CA (140 μM) ( 46 ) are much higher than their K m of SULT2A8, 2.9 μM ( 79 ) and 21.1 μM ( 50 ), respectively. We speculate that most SULT2A8 protein is stabilized by both PAPS and T-CA in hepatocytes, that the high sulfonation efficiency and homeostasis of T-CA can be probably maintained in mouse ( 45 ).…”

“…Interestingly, multiple sequence alignment of all SULTs reveals that a conserved catalytic histidine (His99 in hSULT2A1) is substituted by a leucine in SULT2A8. Although it has been proposed that nonconserved His48 serves as the catalytic residue for the bile acid sulfonation in SULT2A8 ( 50 ), the underlying mechanistic features are not understood. In the present study, we have solved the crystal structure of mSULT2A8 in complex with a cofactor analogue adenosine 3’,5’-diphosphate (PAP) and a 7α-OH substrate CA.…”