Geschmack ist vorhersagbar: Mit FlavorMiner. FlavorMiner heißt das Tool, das IPB-Chemiker und Partner aus Kolumbien jüngst entwickelt haben. Das Programm kann, basierend auf maschinellem Lernen (KI), anhand der…
Seit Februar 2021 bietet Wolfgang Brandt, ehemaliger Leiter der Arbeitsgruppe Computerchemie am IPB, sein Citizen Science-Projekt zur Pilzbestimmung an. Dafür hat er in regelmäßigen Abständen öffentliche Vorträge zur Vielfalt…
Structures of the serine carboxypeptidase‐like enzymes 1‐O ‐sinapoyl‐β‐glucose:l ‐malate sinapoyltransferase (SMT) and 1‐O ‐sinapoyl‐β‐glucose:choline sinapoyltransferase (SCT) were modeled to gain insight into determinants of specificity and substrate recognition. The structures reveal the α/β‐hydrolase fold as scaffold for the catalytic triad Ser‐His‐Asp. The recombinant mutants of SMT Ser173Ala and His411Ala were inactive, whereas Asp358Ala displayed residual activity of 20%. 1‐O ‐sinapoyl‐β‐glucose recognition is mediated by a network of hydrogen bonds. The glucose moiety is recognized by a hydrogen bond network including Trp71, Asn73, Glu87 and Asp172. The conserved Asp172 at the sequence position preceding the catalytic serine meets sterical requirements for the glucose moiety. The mutant Asn73Ala with a residual activity of 13% underscores the importance of the intact hydrogen bond network. Arg322 is of key importance by hydrogen bonding of 1‐O ‐sinapoyl‐β‐glucose and l ‐malate. By conformational change, Arg322 transfers l ‐malate to a position favoring its activation by His411. Accordingly, the mutant Arg322Glu showed 1% residual activity. Glu215 and Arg219 establish hydrogen bonds with the sinapoyl moiety. The backbone amide hydrogens of Gly75 and Tyr174 were shown to form the oxyanion hole, stabilizing the transition state. SCT reveals also the catalytic triad and a hydrogen bond network for 1‐O ‐sinapoyl‐β‐glucose recognition, but Glu274, Glu447, Thr445 and Cys281 are crucial for positioning of choline.
Publikation
Milkowski, C.; Baumert, A.; Strack, D.;Identification of four Arabidopsis genes encoding hydroxycinnamate glucosyltransferasesFEBS Lett.486183-184(2000)DOI: 10.1016/S0014-5793(00)02270-5
Vinblastine and vincristine are two medically important bisindole alkaloids from Catharanthus roseus (Madagascar periwinkle). Attempts at production in cell cultures failed because a part of the complex pathway was not active, i.e. from tabersonine to vindoline. It starts with tabersonine 16-hydroxylase (T16H), a cytochrome P450-dependent enzyme. We now show that T16H is induced in the suspension culture by light and we report the cloning of the cDNA. The enzyme was expressed in Escherichia coli as translational fusion with the P450 reductase from C. roseus, and the reaction product was identified by mass spectrometry. The protein (CYP71D12) shares 47–52% identity with other members of the CYP71D subfamily with unknown function. The induction by light was strongly enhanced by a nutritional downshift (transfer into 8% aqueous sucrose). We discuss the possibility that the entire pathway to bisindoles can be expressed in suspension cultures.