Nicotianamine is an important metal ligand in plants. Surprisingly, recent genome sequencing revealed that ascomycetes encode proteins with similarity to plant nicotianamine synthases (NAS). By expression in a Zn2+‐hypersensitive fission yeast mutant we show for a protein from Neurospora crassa that it indeed possesses NAS activity. Using electrospray‐ionization‐quadrupole‐time‐of‐flight mass spectrometry we prove the formation of nicotianamine in N. crassa . Transcript level is strongly upregulated under Zn deficiency as shown by real‐time PCR. These findings demonstrate that nicotianamine is more widespread in nature than anticipated and provide further evidence for a function of nicotianamine as a cytosolic chelator of Zn2+ ions.

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.

Monochlorobimane was used as a model xenobiotic for Arabidopsis to directly monitor the compartmentation of glutathione‐bimane conjugates in situ and to quantify degradation intermediates in vitro. Vacuolar sequestration of the conjugate was very fast and outcompeted carboxypeptidation to the γ‐glutamylcysteine‐bimane intermediate (γ‐EC‐B) by phytochelatin synthase (PCS) in the cytosol. Following vacuolar sequestration, degradation proceeded to cysteine‐bimane without intermediate. Only co‐infiltration of monochlorobimane with Cd2+ and Cu2+ increased γ‐EC‐B formation to 4% and 25%, respectively, within 60 min. The role of PCS under simultaneous heavy metal stress was confirmed by investigation of different pcs1 null‐mutants. In the absence of elevated heavy metal concentrations glutathione‐conjugates are therefore first sequestered to the vacuole and subsequently degraded with the initial breakdown step being rate‐limiting.