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Two mutants of protocatechuate 3,4-dioxygenase from Acinetobacter sp. ADP1 (Ac 3,4-PCD) have been purified, characterized and their structure determined by x-ray crystallography. These mutants were created through a screening procedure that can select for enzymes with null mutations, temperature sensitive mutations, or even enzymes with altered activities. An Arg457 to Ser mutation was found which led to a null phenotype. Arg457 is postulated to be important for stabilization of the developing carbanion at C4 during catalysis. This carbanion is believed to be critical to the electrophilic attack of C4 by oxygen, which generally is a spin forbidden process. The purified enzyme retains some activity suggesting that Arg457 is not essential to turnover. The crystal structure of the Arg457-> Ser mutant in complex with its natural substrate protocatechuate (PCA) and 4-NC yields interesting insights into the role of Arg457 in facilitating catalysis. A second mutant was obtained by selecting for an enzyme with an increased ability to turn over catechol. A single mutation Arg133-> His led to an Acinetobacter sp. ADP1 which could grow on benzoate using Ac 3,4-PCD as the dioxygenase. Arg133 is a residue close to the entrance of the active site, and was postulated to be involved in either recruitment of positively charged molecules to the active site or as a counter ion to the carboxyl group on protocatechuate. The purified enzyme shows a marked decrease in turnover of PCA and a substantial increase in the turnover of catechol, and therefore has changed its substrate specificity. The structure of this mutation with catechol, PCA, and 4-nitrocatechol yields insight into the nature of these kinetic findings. The structures also suggest a new chelated intial binding step prior to dissociation of Tyr447. |
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