Nucleophile Specificity in Anthranilate Synthase, Aminodeoxychorismate Synthase, Isochorismate Synthase, and Salicylate Synthase

Abstract

Anthranilate synthase (AS), aminodeoxychorismate synthase (ADCS), isochorismate synthase (IS), and salicylate synthase (SS) are structurally homologous chorismate-utilizing enzymes that carry out the first committed step in the formation of tryptophan, folate, and the siderophores enterobactin and mycobactin, respectively. Each enzyme catalyzes a nucleophilic substitution reaction, but IS and SS are uniquely able to employ water as a nucleophile. Lys147 has been proposed to be the catalytic base that activates water for nucleophilic attack in IS and SS reactions; in AS and ADCS, glutamine occupies the analogous position. To probe the role of Lys147 as a catalytic base, the K147Q IS, K147Q SS, Q147K AS, and Q147K ADCS mutants were prepared and enzyme reactions were analyzed by high-performance liquid chromatography. Q147K AS employs water as a nucleophile to a small extent, and the cognate activities of K147Q IS and K147Q SS were reduced approximately 25- and approximately 50-fold, respectively. Therefore, Lys147 is not solely responsible for activation of water as a nucleophile. Additional factors that contribute to water activation are proposed. A change in substrate preference for K147Q SS pyruvate lyase activity indicates Lys147 partially controls SS reaction specificity. Finally, we demonstrate that AS, ADCS, IS, and SS do not possess chorismate mutase promiscuous activity, contrary to several previous reports.

Conversion of Anthranilate Synthase into Isochorismate Synthase: Implications for the Evolution of Chorismate‐Utilizing Enzymes

“…[10] Ziebart and Toney reported in ac omprehensive study on nucleophile specificity in MST enzymes that the Gln263Lys variant of stAS (equivalent to our KML variant) formed traces of IC and SA. [11] However,o nly 0.008 %a nd 0.03 %o fC H were converted to IC and SA, respectively,u nder conditions comparable to our experimental setup.Inany case,the central finding of our work, namely inversion of the nucleophile specificity of stAS through af ew amino acid replacements,a se xemplified by the KAS and KAA variants,isindependent of whether the single Gln263Lys replacement leads to no or extremely low amounts of IC or SA.…”

Salicylic Acid and Defense Responses in Plants

“…As expected, ICS1 contains a putative plastid transit sequence and a cleavage site (Wildermuth et al 2001 ). The high affi nity of ICS1 for chorismate allows ICS1 to compete successfully with other pathogen-induced enzymes that use chorismate as their substrate, such as anthranilate synthase (Strawn et al 2007 ;Ziebart and Toney 2010 ). Unlike the bifunctional SAS, the recombinant ICS1 only converts chorismate to IC, since no SA was detected in the products of this reaction (Strawn et al 2007 ).…”

Umwandlung einer Anthranilatsynthase in eine Isochorismatsynthase: Implikationen für die Evolution von Chorismat‐umsetzenden Enzymen

“…[11] Zunächst wurde angenommen, dass zwei Wasserstoffbrückenakzeptoren die Deprotonierung der e-Aminogruppe von Lys263 unterstützen. Allerdings ist einer dieser Akzeptoren, ein Glutamat, nicht nur in …”

“…[10] Dahingegen berichteten Ziebart und Toney in einer umfassenden Studie zur Nukleophilspezifität in MST-Enzymen, dass der einfache Gln263Lys-Austausch in stAS (entsprechend unserer KMLVa riante) zur Bildung von Spuren von IC und SA führt. [11] Allerdings wurden von der entsprechenden stAS-Variante unter experimentellen Bedingungen, die mit unserem Ansatz vergleichbar sind, nur 0.008 %b zw.0 .03 %d es eingesetzten CH zu IC bzw.S Au mgesetzt. Die Frage,o bd er einfache Gln263Lys-Austausch zu keiner ICS/SS-Aktivität oder zu Bildung von extrem geringen Mengen IC und SA führt, hat jedoch keinen Einfluss auf das zentrale Ergebnis unserer Arbeit:die Inversion der Nukleophilspezifität der stAS durch wenige Aminosäureaustausche,v erdeutlicht durch die KASund KAA-Varianten.…”