Abstract
Ca(2+) is essential for in vitro activity of Erwinia chrysanthemi pectate lyase C (PelC). Crystallographic analyses of 11 PelC-Ca(2+) complexes, formed at pH 4.5, 9.5, and 11.2 under varying Ca(2+) concentrations, have been solved and refined at a resolution of 2.2 A. The Ca(2+) site represents a new motif for Ca(2+), consisting primarily of beta-turns and beta-strands. The principal differences between PelC and the PelC-Ca(2+) structures at all pH values are the side-chain conformations of Asp-129 and Glu-166 as well as the occupancies of four water molecules. According to calculations of pK(a) values, the presence of Ca(2+) and associated structural changes lower the pK(a) of Arg-218, the amino acid responsible for proton abstraction during catalysis. The Ca(2+) affinity for PelC is weak, as the K(d) was estimated to be 0.132 (+/-0.004) mm at pH 9.5, 1.09 (+/-0.29) mm at pH 11.2, and 5.84 (+/-0.41) mm at pH 4.5 from x-ray diffraction studies and 0.133 (+/-0.045) mm at pH 9.5 from intrinsic tryptophan fluorescence measurements. Given the pH dependence of Ca(2+) affinity, PelC activity at pH 4.5 has been reexamined. At saturating Ca(2+) concentrations, PelC activity increases 10-fold at pH 4.5 but is less than 1% of maximal activity at pH 9.5. Taken together, the studies suggest that the primary Ca(2+) ion in PelC has multiple functions.
Highlights
Erwinia sp. are pathogenic bacteria that are causally involved in soft rot diseases in a wide variety of plant crops [1, 2]
Are all Ca2ϩ ions essential for in vitro catalytic activity? Are the Ca2ϩ ions only required for substrate binding, or do any have an active catalytic role? If so, which Ca2ϩ ion has an active catalytic role, and how might the ion participate in catalysis? Is the primary Ca2ϩ ion involved in the polygalacturonic acid hydrolase activity that is observed in some pectate lyases [18, 19]? In view of the observations that the pH of the plant cell wall increases as the pectate component is degraded [20], how does pH affect the affinity of the Ca2ϩ ions for pectate lyase C (PelC)? To address these questions, various studies with Ca2ϩ have been initiated
The results presented explore the binding of the primary Ca2ϩ ion to PelC over a broad pH range
Summary
Erwinia sp. are pathogenic bacteria that are causally involved in soft rot diseases in a wide variety of plant crops [1, 2]. Is the primary Ca2ϩ ion involved in the polygalacturonic acid hydrolase activity that is observed in some pectate lyases [18, 19]? In view of the observations that the pH of the plant cell wall increases as the pectate component is degraded [20], how does pH affect the affinity of the Ca2ϩ ions for PelC?
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