Skip to main content
. 2014 May 22;289(27):18719–18735. doi: 10.1074/jbc.M114.575076

TABLE 2.

Site-directed mutations generated in E. coli UppP enzyme

ND is not determined. E137A is expressed at levels too low to obtain reliable phosphatase activity data. The P- and C-sites indicate periplasmic and cytoplasmic sites, respectively. TM indicates transmembrane helix.

UppP mutants Predicted localization Relative activities of the wild type Suggested role
%
Region I
    E17A Active site (P-site) 26 H-bond with pyrophosphate of UppP via the Mg2+
    E21A Active site (P-site) 40 H-bond with pyrophosphate of UppP via the Mg2+
    E17A/E21A Active site (P-site) <1 H-bond with pyrophosphate of UppP via the Mg2+
    H30A Active site (P-site) <1 Initiating a nucleophilic attack on the phosphorus center

Region II (structural P-loop)
    S173A Active site (P-site) <1 The backbone oxygen H-bond with Arg-174
    R174A Active site (P-site) <1 H-bond with oxygen atom of α-phosphate of UppP
    S175A Active site (P-site) 32 Interaction with phosphate group of UppP
    T178A Active site (P-site) <1 H-bond with the backbone NH of Arg-174

Other mutants
    E41A Aqueous interface (P-site) 85 Loop structural maintenance
    D43A Aqueous interface (P-site) 73 Loop structural maintenance
    E49A Membrane-water interface (P-site) 36 TM2 structural maintenance
    Q53A Membrane-water interface (P-site) 14 TM2 structural maintenance
    D111A Membrane-water interface (P-site) 100
    E137A At the end of TM4 (C-site) ND Salt bridge with Arg-67 and His-94
    D150A Membrane-water interface (C-site) 64
    R189A Aqueous interface (P-site) 11 Stabilizing His-30
    E194A Aqueous interface (P-site) 31 Stabilizing Arg-189
    R261A At the end of TM8 (P-site) <1 H-bond with Ser-173