Identification of functionally important amino-terminal arginines of Agrobacterium tumefaciens ADP-glucose pyrophosphorylase by alanine scanning mutagenesis.

Treatment of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase with the arginyl reagent phenylglyoxal resulted in complete desensitization to fructose 6-phosphate (F6P) activation, and partial desensitization to pyruvate activation. The enzyme was protected from desensitization by ATP, F6P, pyruvate, and phosphate. Alignment studies revealed that this enzyme contains arginine residues in the amino-terminal region that are relatively conserved in similarly regulated ADP-glucose pyrophosphorylases. To functionally evaluate the role(s) of these arginines, alanine scanning mutagenesis was performed to generate the following enzymes: R5A, R11A, R22A, R25A, R32A, R33A, R45A, and R60A. All of the enzymes, except R60A, were successfully expressed and purified to near homogeneity. Both the R5A and R11A enzymes displayed desensitization to pyruvate, partial activation by F6P, and increased sensitivity to phosphate inhibition. Both the R22A and R25A enzymes exhibited reduced V(max) values in the absence of activators, lower apparent affinities for ATP and F6P, and reduced sensitivities to phosphate. The presence of F6P restored R22A enzyme activity, while the R25A enzyme exhibited only approximately 1.5% of the wild-type activity. The R32A enzyme displayed an approximately 11.5-fold reduced affinity for F6P while exhibiting behavior identical to that of the wild type with respect to pyruvate activation. Both the R33A and R45A enzymes demonstrated a higher activity than the wild-type enzyme in the absence of activators, no response to F6P, partial activation by pyruvate, and desensitization to phosphate inhibition. These altered enzymes were also insensitive to phenylglyoxal. The data demonstrate unique functional roles for these arginines and the presence of separate subsites for the activators.

Cloning and sequencing of glycogen metabolism genes from Rhodobacter sphaeroides 2.4.1. Expression and characterization of recombinant ADP-glucose pyrophosphorylase.

A 6-kb DNA fragment of the Rhodobacter sphaeroides 2.4.1 glg operon was cloned from a genomic library using a polymerase chain reaction probe coding for part of the ADP-glucose pyrophosphorylase (glgC) gene. The DNA fragment was sequenced and found to harbor complete open reading frames for the glgC and glgA (glycogen synthase) genes and partial sequences corresponding to glgP (glycogen phosphorylase) and glgX (glucan hydrolase/transferase) genes. The genomic fragment also contained an apparent truncated sequence corresponding to the C-terminus of the glgB gene (branching enzyme). The presence of active branching enzyme activity in crude sonicates of Rb. sphaeroides cells indicates that the genome contains a full-length glgB at another location. The structure of this operon in relation to other glg operons is further discussed. The deduced sequence of the ADP-glucose pyrophosphorylase enzyme is compared to other known ADP-glucose pyrophosphorylase sequences and discussed in relation to the allosteric regulation of this enzyme family. The glgC gene was subcloned in the vector pSE420 (Invitrogen) for high-level expression in E. coli. The successful overexpression of the recombinant enzyme allowed for the purification of over 35 mg of protein from 10 g of cells, representing a dramatic improvement over enzyme isolation from the native strain. The recombinant enzyme was purified to near homogeneity and found to be physically, immunologically, and kinetically identical to the native enzyme, verifying the fidelity of the cloning step.

Designing zinc-finger ADR1 mutants with altered specificity of DNA binding to T in UAS1 sequences.

Yeast ADR1 contains two Cys2,His2 zinc fingers needed for DNA binding to the upstream activation sequence UAS1, with bases T5T6G7-G8A9G10 in the ADH2 promoter. Potential DNA-contacting amino acid residues at -1, +3, and +6 in the alpha-helical domains of ADR1's fingers one and two include RHR-RLR; however, the latter finger two residues Leu146 and Arg149 had not proved to be crucial for ADR1 binding, even though Leu146-T6 and Arg149-T5 interactions with UAS1 DNA were predicted. We altered Leu146 or Arg149 by PCR cassette mutagenesis, to study ADR1 mutant binding to 16 UAS1 variants of thymine bases T5 and T6. Mutation of Leu146 to His, making finger two (RLR) like finger one (RHR), decreased binding to wild type UAS1 having T6, but enhanced its binding strength to sequences having purines G6 or A6, similar to binding seen between finger one's His118 and base A9 of UAS1. Mutating Leu146 to Lys caused this finger two RKR mutant to bind strongly to both G6 and T6, possibly by lysine's amine H-bonding to the carbonyl of guanine or thymine. Specificity of ADR1 for UAS1 with T6 may thus be due to hydrophobic interaction between Leu146 and the T6 methyl group. ADR1 mutants with either His or Lys in the central +3 residue (146) of zinc finger two, which have Arg149 in the +6 alpha-helical position, bind with UAS1 mutant sequences having G5 very strongly, T5 strongly, A5 intermediately, and C5 weakly.(ABSTRACT TRUNCATED AT 250 WORDS)