Minimale Umgestaltung aktiver Enzymtaschen – wie man alten Enzymen neue Kunststücke beibringt

Abstract

Während die Natur ihre Katalysatoren über Millionen von Jahren entwickelt hat, versuchen Enzymingenieure, dies ein wenig schneller zu bewerkstelligen. Die aktive Tasche eines Enzyms bildet eine stark optimierte Mikroumgebung für die Katalyse chemischer Transformationen in biologischen Systemen – folglich können Änderungen in diesen Zentren die Enzymaktivität stark beeinflussen. Daher bietet die Voraussage und Kontrolle solcher Effekte einen vielversprechenden Weg, um zu neuen Funktionen zu gelangen. Der Ansatz, bei dem minimale Modifikationen in den aktiven Taschen von Enzymen (z. B. durch gezielte Mutagenese und Hinzufügen neuer reaktiver Funktionalitäten) deren katalytisches Repertoire erweitern sollen, ist der Schwerpunkt dieses Aufsatzes. Dabei kann eine neue Aktivität oft schon durch eine einzige Mutation erreicht werden. Die vielen erfolgreichen Beispiele für die Umgestaltung von aktiven Taschen durch minimale Mutationen geben wertvolle Einblicke in die Evolution von Enzymen und bahnen neue Wege in der Erforschung der Biokatalysatoren.

Ostensible Enzyme Promiscuity: Alkene Cleavage by Peroxidases

“…[2,5] Furthermore, promiscuous activity was subdivided into three groups, namely 1) condition-, 2) substrate-and 3) catalyticpromiscuity. [6,7] In a recent article entitled How enzymes work, [8] it was stated that the tasks of an enzyme are 1) to bring the reacting species together in a geometry that favours the reaction, 2) to distort the substrate and hence to stabilise one substrate conformation better than the others and to permit it to follow a specific reaction mechanism and 3) to create an inner microenvironments in the protein core leading to altered pK a s of the involved amino acid residues. All these apects contribute to the enzymatic catalysis.…”

Suppressed Native Hydrolytic Activity of a Lipase to Reveal Promiscuous Michael Addition Activity in Water

“…Over the past few years, there has been significant progress in research related to enzyme catalytic promiscuity, [4][5][6][7][8] that is, the capacity of enzymes to catalyze reactions other than the ones for which they are evolved. Promiscuous activities can provide clues on how to modify enzymes or reaction conditions to enhance desired qualities or suppress unwanted activities.…”

Current Trends in Asymmetric Synthesis with Aldolases

“…[21,[60][61][62][63] 2-Keto-3-deoxygluconate (KDGlu) aldolase from the hyperthermophile Sulfolobus solfataricus is a class I pyruvate aldolase with broad acceptor specificity in-cluding non-phosphorylated aldehydes, but, as mentioned above, lacks stereoselectivity for conformationally free substrates; for example, the aldol addition of pyruvate to d-glyceraldehyde gave a 55:45 mixture of d-2-keto-3-deoxygluconate (34) and d-2-keto-3-deoxygalactonate (35) (Scheme 10). [21,[60][61][62][63] 2-Keto-3-deoxygluconate (KDGlu) aldolase from the hyperthermophile Sulfolobus solfataricus is a class I pyruvate aldolase with broad acceptor specificity in-cluding non-phosphorylated aldehydes, but, as mentioned above, lacks stereoselectivity for conformationally free substrates; for example, the aldol addition of pyruvate to d-glyceraldehyde gave a 55:45 mixture of d-2-keto-3-deoxygluconate (34) and d-2-keto-3-deoxygalactonate (35) (Scheme 10).…”

“…[21,22] The directed evolution approach for the alteration of enzymatic activity towards a specific target does not require any structural or mechanistic knowledge of the enzyme. [21,22] The directed evolution approach for the alteration of enzymatic activity towards a specific target does not require any structural or mechanistic knowledge of the enzyme.…”