Glycerol-3-phosphate Cytidylyltransferase: STRUCTURAL CHANGES INDUCED BY BINDING OF CDP-GLYCEROL AND THE ROLE OF LYSINE RESIDUES IN CATALYSIS

Katherine A Pattridge,Christian H Weber,Jon A Friesen,Subramaniam Sanker,Claudia Kent,Martha L Ludwig

doi:10.1074/jbc.m306174200

Katherine A Pattridge, Christian H Weber + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m306174200

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Abstract

The bacterial enzyme, glycerol-3-phosphate cytidylyltransferase (GCT), is a model for mammalian cytidylyltransferases and is a member of a large superfamily of nucleotidyltransferases. Dimeric GCT from Bacillus subtilis displays unusual negative cooperativity in substrate binding and appears to form products only when both active sites are occupied by substrates. Here we describe a complex of GCT with the product, CDP-glycerol, in a crystal structure in which bound sulfate serves as a partial mimic of the second product, pyrophosphate. Binding of sulfate to form a pseudo-ternary complex is observed in three of the four chains constituting the asymmetric unit and is accompanied by a backbone rearrangement at Asp11 and ordering of the C-terminal helix. Comparison with the CTP complex of GCT, determined previously, reveals that in the product complex the active site closes around the glycerol phosphate moiety with a concerted motion of the segment 37-47 that includes helix B. This rearrangement allows lysines 44 and 46 to interact with the glycerol and cytosine phosphates of CDP-glycerol. Binding of CDP-glycerol also induces smaller movements of residues 92-100. Roles of lysines 44 and 46 in catalysis have been confirmed by mutagenesis of these residues to alanine, which decreases Vmax(app) and has profound effects on the Km(app) for glycerol-3-phosphate.

Highlights

The bacterial enzyme, glycerol-3-phosphate cytidylyltransferase (GCT), is a model for mammalian cytidylyltransferases and is a member of a large superfamily of nucleotidyltransferases
We describe a complex of GCT with the product, CDP-glycerol, in a crystal structure in which bound sulfate serves as a partial mimic of the second product, pyrophosphate
The wild-type protein used for crystallization was expressed and purified by chromatography on Blue-Sepharose (Amersham Biosciences) essentially as originally described for GCT expressed in E. coli [15] except that elution from Blue-Sepharose was accomplished with 100 mM phosphate instead of CTP

Summary

EXPERIMENTAL PROCEDURES

Materials—CTP, glycerol-3-phosphate, protease inhibitors, and imidazole were from Sigma. PCR reaction #1 amplified 5Ј to the Lys or Lys codon. PCR reaction #2 amplified 3Ј to the Lys or Lys codon. After centrifugation at 40,000 ϫ g for 1 h, the soluble lysate was applied to a 5-ml column of nickelnitrilotriacetic acid resin equilibrated with 10 mM Tris, pH 8.0, 300 mM NaCl, 10% glycerol. The column was washed with 20 mM Tris, pH 8.0, 300 mM NaCl, 10% glycerol, 20 mM galactose, 50 mM imidazole. The wild-type protein used for crystallization was expressed and purified by chromatography on Blue-Sepharose (Amersham Biosciences) essentially as originally described for GCT expressed in E. coli [15] except that elution from Blue-Sepharose was accomplished with 100 mM phosphate instead of CTP. Substrate dependence curves were fit to the Michaelis-Menten equation v ϭ (Vmax * [S])/(Km ϩ [S]) or the Hill equation v ϭ (Vmax * [S]n)/(KЈ ϩ [S]n) using Kaleidograph (Synergy software) to derive

TABLE II Model refinement statistics

RESULTS

TABLE III Ligand interactions

TABLE IV Kinetic values for lysine mutants

DISCUSSION