Combinatorial libraries of synthetic and natural products are an important source of molecular information for the interrogation of biological targets. Methods for the intracellular production of libraries of small, stable molecules would be a valuable addition to existing library technologies by combining the discovery potential inherent in small molecules with the large library sizes that can be realized by intracellular methods. We have explored the use of split inteins (internal proteins) for the intracellular catalysis of peptide backbone cyclization as a method for generating proteins and small peptides that are stabilized against cellular catabolism. The DnaE split intein from Synechocystis sp. PCC6803 was used to cyclize the Escherichia coli enzyme dihydrofolate reductase and to produce the cyclic, eight-amino acid tyrosinase inhibitor pseudostellarin F in bacteria. Cyclic dihydrofolate reductase displayed improved in vitro thermostability, and pseudostellarin F production was readily apparent in vivo through its inhibition of melanin production catalyzed by recombinant Streptomyces antibioticus tyrosinase. The ability to generate and screen for backbone cyclic products in vivo is an important milestone toward the goal of generating intracellular cyclic peptide and protein libraries.

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As a continuation to our studies on the importance of interloop interactions in the Escherichia coli DHFR catalytic cycle, we have investigated the role of the betaG-betaH loop in modulating the closed and occluded conformations of the Met20 loop during the DHFR catalytic cycle. Specifically, to assess the importance of the hydrogen bond formed between Ser148 in the betaG-betaH loop and the Met20 loop, Ser148 was independently substituted with aspartic acid, alanine, and lysine. Moreover, the betaG-betaH loop was deleted entirely to yield the Delta(146-148) DHFR mutant. Steady-state turnover rates for all mutants were at most 3-fold lower than the wild-type rate. Lack of an isotope effect on this rate indicated the chemistry step does not contribute to the steady-state turnover. Consistent with this finding, hydride transfer rates for the DHFR mutants were at least 10-fold greater than the observed steady-state rates. The values ranged from a 30% decrease (Ser148Ala and Ser148Lys) to a 50% increase (Ser148Asp) in rate relative to that of the wild type. Modifications of the betaG-betaH loop enhanced the affinity for the cofactor and decreased the affinity for pterin, as determined by the K(D) values of the mutant proteins. Further analysis of Ser148Ala and Delta(146-148) DHFRs indicated these effects were manifest mainly in ligand off rates, although in some cases the on rate was affected. The Ser148Asp and Delta(146-148) mutations perturbed the preferred catalytic cycle through the introduction of branching at key intermediates. Rather than following the single WT pathway which involves loss of NADP(+) and rebinding of NADPH to precede loss of the product H4F (negative cooperativity), the mutants can reenter the catalytic cycle through different pathways. These findings suggest that the role of the interloop interaction between the betaG-betaH loop and the Met20 loop is to modulate ligand off rates allowing for proper cycling through the preferred kinetic pathway.

Metal ions play enormously important roles in both enzymic and nonenzymic"] hydrolysis of phosphate diesters including DNA and RNA. Some enzymes such as 3'4' exonuclease from the Klenow fragment of DNA polymerase I, ['] ['] are activated by two or more metal ions. Functional models of phosphoesterases with two metal atoms in the active site may provide mechanistic insights into the role of the metal ions in the enzymes. We have shown that dinuclear copper(I1) complexes, in which both Cu atoms reversibly coordinate the two phosphoryl oxygens of a bridging phosphate diester simultaneously, can be highly reactive for cleaving the ester."] Here we report on the hydrolysis of a phosphate diester that is doubly coordinated to a dinuclear cobalt(1n) complex. Since this complex does not undergo rapid substitution. it is possible to examine the effect of the dinuclear complex alone on the hydrolysis of the coordinated phosphate diester.The perchlorate salts of the dinuclear cobalt(n1) complex cations 1 and 2['01 with bridging dimethyl phosphate and bridging methyl p-nitrophenyl phosphate ligands, respectively, were synthesized by treating [([9]aneN,),Co,(OH),](NO,), ([9]aneN3 = 1,4,7-triazacyclononane) (25 mM) with the sodium salt of the phosphate diester (25 mM) in aqueous HCIO, (1 M). ["] 31P N M R spectroscopy is a useful method for characterizing the bridging phosphate diester in 1 and 2.[L01 Monodentate coordination of dimethyl phosphate to mononuclear Co"' complexes results in a downfield shift of the phosphorus signal by about 7ppm.["] The phosphorus signals of the bridging phosphates in 1 and 2 are shifted about 14 ppm downfield relative to the signals of the corresponding free phosphates. Over the years many interesting dinuclear metal complexes structurally related to 1 and 2 that contain bridging carboxylate" 'I rather than bridging phosphate groups have been reported. Many structures of substitutionally labile dinuclear metal complexes with bridging phosphate diesters have also been reported. [9, 13] Figure 1 shows the crystal structure of trication l. [14] This is the first structure of a complex in which a phosphate diester ligand bridges two substitutionally inert metal centers.The distance between the two cobalt(iI1) centers in 1 (2.9 A) is somewhat shorter than intermetallic distances in dinuclear phosphodiesterases (ca. 4 A)[2, but comparable to that recently reported for a purple acid phosphatase (ca. 3.1 A).[7d1 The distances between the phosphorus and the bridging oxygen atoms (PI -05, PI -06 3.230(7) A) are slightly shorter than the P -0 van der Waals distance (3.3 A).

A cis-diaqua copper(i1) complex has been shown to promote the transesterification of a phosphate diester with second-order dependence on the metal complex.