Figure 3.
Reduction of the Cys194–Cys200 disulfide bond inactivates
and partially disorders CcmM209. (A) Effect of 10 mM TCEP on the
catalytic activity of CcmM209 (0.02 mg·mL^-1) and
CcmMχ173 (0.01 mg·mL^-1). CcmMχ173 is constitutively
active under both oxidizing and reducing (10 mM TCEP)
conditions, whereas CcmM209 is active under oxidizing
conditions, but activity is reduced to uncatalyzed rates in the
presence of 10 mM TCEP. The CcmM209C200S variant is inactive
under both oxidizing and reducing conditions. Assay condition:
200 mM EPPS/NaOH, pH 7.8, 1 mM NaCl, 600 μM at
30 °C. (B) Tryptophan fluorescence of CcmM193, CcmM209,
and CcmMχ173 under oxidizing and reducing (10 mM DTT)
conditions. The constitutively active CcmMχ173 chimera is
strongly fluorescent under both conditions, the constitutively
inactive construct CcmM193 is more weakly fluorescent under both
conditions, while the CcmM209 switches from strongly fluorescent
under oxidizing, to weakly fluorescent under reducing
conditions. This is consistent with the quenching of W13 caused
by the unstructuring of the β1–β2 loop and the α2 and α3
helices upon reduction of the C194–C200 disulfide bond. (C) E.
coli’s cytosolic reducing machinery keeps CcmM209 in an
inactive state. Activity traces for fresh cell lysate containing
over expressed CcmM209 in the absence and presence (25 mM) of
the thiol oxidizing agent diamide. Activation of CcmM209 also
occurs during the purification process due to oxidation of the
protein by molecular O[2]. Cell lysate containing the
CcmM209C200S variant remains inactive in the presence of
diamide, indicating that the activation mechanism is dependent
upon the potential to form a C194–C200 disulfide bond. (D)
Details showing the regions of the β1–β2 loop and α2–α3
helices that are necessary for stabilizing the protein in an
active conformation. (E) Excerpt of an alignment of CcmM
sequences. Species are abbreviated as Te,Thermosynechococcus
elongatus-BP1; Gv, Gloeobacter violaceus PCC 7421; Tric,
Trichodesmium erythraeum IMS101; Np, Nostoc punctiforme PCC
73102; Cw, Crocosphaera watsonii WH 8501; 6803, Synechocystis
sp. PCC 6803; 7942, Synechococcus elongatus PCC 7942; 7335,
Synechococcus sp. PCC 7335. Among the shown species, Te, Gv, and
Tric contain no ccaA homolog; the other species shown do. The
sequences for 6803, 7942, and 7335 do not conserve C194, C200
(green ellipses), the β1–β2 loop (cyan box with W13 marked
with a cyan ellipse), or critical elements in the α2 and α3
helices (orange box, N184 magenta ellipse). Consequently, they
(along with six other species not depicted) are unlikely to show
CA activity.
at
30 °C. (B) Tryptophan fluorescence of CcmM193, CcmM209,
and CcmMχ173 under oxidizing and reducing (10 mM DTT)
conditions. The constitutively active CcmMχ173 chimera is
strongly fluorescent under both conditions, the constitutively
inactive construct CcmM193 is more weakly fluorescent under both
conditions, while the CcmM209 switches from strongly fluorescent
under oxidizing, to weakly fluorescent under reducing
conditions. This is consistent with the quenching of W13 caused
by the unstructuring of the β1–β2 loop and the α2 and α3
helices upon reduction of the C194–C200 disulfide bond. (C) E.
coli’s cytosolic reducing machinery keeps CcmM209 in an
inactive state. Activity traces for fresh cell lysate containing
over expressed CcmM209 in the absence and presence (25 mM) of
the thiol oxidizing agent diamide. Activation of CcmM209 also
occurs during the purification process due to oxidation of the
protein by molecular O[2]. Cell lysate containing the
CcmM209C200S variant remains inactive in the presence of
diamide, indicating that the activation mechanism is dependent
upon the potential to form a C194–C200 disulfide bond. (D)
Details showing the regions of the β1–β2 loop and α2–α3
helices that are necessary for stabilizing the protein in an
active conformation. (E) Excerpt of an alignment of CcmM
sequences. Species are abbreviated as Te,Thermosynechococcus
elongatus-BP1; Gv, Gloeobacter violaceus PCC 7421; Tric,
Trichodesmium erythraeum IMS101; Np, Nostoc punctiforme PCC
73102; Cw, Crocosphaera watsonii WH 8501; 6803, Synechocystis
sp. PCC 6803; 7942, Synechococcus elongatus PCC 7942; 7335,
Synechococcus sp. PCC 7335. Among the shown species, Te, Gv, and
Tric contain no ccaA homolog; the other species shown do. The
sequences for 6803, 7942, and 7335 do not conserve C194, C200
(green ellipses), the β1–β2 loop (cyan box with W13 marked
with a cyan ellipse), or critical elements in the α2 and α3
helices (orange box, N184 magenta ellipse). Consequently, they
(along with six other species not depicted) are unlikely to show
CA activity.