TABLE 1.

Functional assignment of N-sulfamidase active site residues

Amino acids listed in this table were identified by molecular interaction analysis of the N-sulfamidase model depicted in Fig. 3 as well as the proposed enzymatic cleavage mechanism shown in Fig. 4.

Active site residues	Proposed functional role
Cys-80	The active site cysteine is modified into the hydrated form of the FGly-O-γ1 (20) and participates in nucleophilic attack on the sulfate group
Arg-84	This primarily stabilizes the hydrated FGly by interaction with O-γ2. This is also well positioned for proton abstraction from O-γ2 after the catalytic process for elimination of sulfate and regeneration of diol
Asp-159	The negatively charged carboxylate groups of these residues are likely to abstract the proton from the NH3+ group of Lys-160
Lys-160	Upon loss of the proton to Asp-159, the nitrogen of this lysine residue is likely to be sulfated by the NHSO3− group, thereby breaking the existing nitrogen-sulfur bond. This process is mediated by the calcium ion. After this step, the sulfated lysine will be desulfated by the FGly residue. This lysine is also likely to increase electrophilicity of sulfur center by coordinating with the oxygen atoms of the sulfate group
Asp-246, Asn-247, Asp-40, Asn-41	Asp-246, Asp-247, Asp-40, and Asn-41 are well positioned to coordinate with the divalent Ca2+ metal ion. The calcium ion is likely to play the crucial role of coordinating with the nitrogen of NHSO3− group, hence helping Lys-160 to break the N–S bond successfully. Hence, this tetrad is important for optimal N-sulfamidase function. Furthermore, Asp-40 is also understood to donate a proton and enhance nucleophilicity of O-γ1