Structural and Mechanistic Analysis of the Choline Sulfatase from Sinorhizobium melliloti: A Class I Sulfatase Specific for an Alkyl Sulfate Ester

Abstract

Hydrolysis of organic sulfate esters proceeds by two distinct mechanisms, water attacking at either sulfur (S–O bond cleavage) or carbon (C–O bond cleavage). In primary and secondary alkyl sulfates, attack at carbon is favored, whereas in aromatic sulfates and sulfated sugars, attack at sulfur is preferred. This mechanistic distinction is mirrored in the classification of enzymes that catalyze sulfate ester hydrolysis: arylsulfatases (ASs) catalyze S–O cleavage in sulfate sugars and arylsulfates, and alkyl sulfatases break the C–O bond of alkyl sulfates. Sinorhizobium meliloti choline sulfatase (SmCS) efficiently catalyzes the hydrolysis of alkyl sulfate choline-O-sulfate (kcat/KM = 4.8 × 103 s− 1 M− 1) as well as arylsulfate 4-nitrophenyl sulfate (kcat/KM = 12 s− 1 M− 1). Its 2.8-Å resolution X-ray structure shows a buried, largely hydrophobic active site in which a conserved glutamate (Glu386) plays a role in recognition of the quaternary ammonium group of the choline substrate. SmCS structurally resembles members of the alkaline phosphatase superfamily, being most closely related to dimeric ASs and tetrameric phosphonate monoester hydrolases. Although > 70% of the amino acids between protomers align structurally (RMSDs 1.79–1.99 Å), the oligomeric structures show distinctly different packing and protomer–protomer interfaces. The latter also play an important role in active site formation. Mutagenesis of the conserved active site residues typical for ASs, H218O-labeling studies and the observation of catalytically promiscuous behavior toward phosphoesters confirm the close relation to alkaline phosphatase superfamily members and suggest that SmCS is an AS that catalyzes S–O cleavage in alkyl sulfate esters with extreme catalytic proficiency.

High resolution crystal structure of the catalytic domain of MCR-1

“…Several structural homologues from hydrolase superfamily are identified via online DALI server39 including lipooligosaccharide phosphoethanolamine transferase A from Neisseria gonorrhoeae (LptA, PDB: 4KAV and 4KAY)33, phosphoethanolamine transferase C from Campylobacter jejuni (EptC, PDB: 4TN0)34, choline sulfatase (betC, PDB: 4UG4)40 and phosphonate monoester hydrolase (PMH, PDB: 2W8S)41. The nearest structural homologues are LptA and EptC with sequence identities of 40–41% whereas betC and PMH are distant homologues having sequence identities of 16% and 13%, respectively.…”

Sated by a Zero-Calorie Sweetener: Wastewater Bacteria Can Feed on Acesulfame

“…The enzymes involved in the proposed steps might be sulfohydrolases, hydratases, or amidohydrolases. Promiscuous sulfohydrolases attacking aryl as well as alkyl sulfates via S-O bond cleavage can be found within the alkaline phosphatase superfamily (van Loo et al, 2018). The hydrolysis product ANSA could also be formed by the Michael addition of water to the C〓C system catalyzed by various hydratases (Resch and Hanefeld, 2015).…”

Balancing Specificity and Promiscuity in Enzyme Evolution: Multidimensional Activity Transitions in the Alkaline Phosphatase Superfamily

“…Trees based on multiple sequence alignments generated with other methods (Figure S2-S4) and a tree built using Bayesian maximum likelihood ( Figure S5) all support the latter two observations. (B) Expansion of the phylogenetic clade indicated in panel (A) with putative 'new' ASs and PMHs, rooted with an expanded clade of choline sulfatases (represented as CSs), as described previously 54 ). The average sequence identity was 51±12% and 49±18% within the AS and PMH clade respectively (average overall sequence identity between ASs and PMHs is 38±16%).…”

“…The final alignment was done with 85 (putative) PMHs, 95 (putative) ASs (for details see Table S6-S7) and 87 (putative) CSs using the 3D-coffee mode of T-coffee as described previously. 54 These alignments were used as input to build a maximum likelihood phylogenetic tree with RAxML BlackBox 8.0.24, 50 using the Le & Gascuel 55 amino acid substitution matrix (LG) with a g-model for rate heterogeneity, estimate proportion of invariable sites and empirical base frequencies (LG+G+I+F) running on the CIPRES Science Gateway 51 (www.phylo.org/portal2/). Several additional methods (Clustal W, MAFFT or MUSCLE) were employed to create alternative phylogenetic trees, based on the same data and using the same settings ( Figure S8).…”