Formation of phenylalanylserine and cyclo-phenylalanylseryl by protoplasts of Gliocladium virens

GliP, a Multimodular Nonribosomal Peptide Synthetase in Aspergillus fumigatus, Makes the Diketopiperazine Scaffold of Gliotoxin

“…Analysis of Intermediates Tethered to GliP. Each GliP enzyme (10 µM) included in the 120 µL reaction was primed by incubation with 75 mM Tris, pH 7.5, 5 mM MgCl 2 , 300 µM CoASH, and 3 µM Sfp at room temperature for 1 h. Amino acid loading and condensation were then assayed by initiation with 37.5 mM Tris, pH 7.5, 5 mM ATP, and 400 µM of each amino acid included (both 14 C radiolabeled and unlabeled). After 1.5 h, reactions were quenched in 480 µL of 10% TCA, pelleted by centrifugation, and washed with 3 × 100 µL of 10% TCA.…”

“…The reaction was then quenched by acidifying with 20 µL of 50% TCA and centrifuged to pellet the protein. The supernatant containing the released products was analyzed by HPLC on a Vydac 250 × 4.6 mm C18 small pore column, using a 0-18% acetonitrile gradient over 18 min, then holding for an additional 10 min, starting in 0.1% TFA in H 2 O, and monitoring 14 C radioactive counts and absorbance at 260 nm.…”

“…Feeding experiments have demonstrated that the core DKP is biosynthesized from phenylalanine (10) and serine (11); however, there has been some debate as to whether cyclo-(L-phenylalanyl-L-seryl) is an intermediate on the pathway to formation of gliotoxin. In support of such an intermediate, labeled Phe-Ser DKP has been efficiently incorporated into gliotoxin produced by Trichoderma Viride (12,13), and protoplasts of Gliocladium Virens can synthesize the dipeptide phenylalanylserine and the DKP cyclo-phenylalanylseryl (14). Contrarily, the mycelium of Penicillium terlikowskii poorly incorporates cyclo-phenylalanylseryl despite efficient uptake of the DKP (15).…”

“…Contrarily, the mycelium of Penicillium terlikowskii poorly incorporates cyclo-phenylalanylseryl despite efficient uptake of the DKP (15). Furthermore, the protoplasts of G. Virens which make the DKP do not biosynthesize gliotoxin (14), and an "intermediate trapping" experiment in T. Viride showed that the cyclo-dipeptide may only be present in the organism in miniscule amounts under normal growth conditions (13). No other intermediates have been isolated on route to formation of gliotoxin.…”

The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

“…Until recently, the mechanism of gliotoxin biosynthesis had proven to be especially refractory to analysis, despite multiple studies between 1960 and 1985 involving radiolabeled precursor feeding and analysis of proteins produced during gliotoxin biosynthesis (2,18). Since the discovery of the gliotoxin gene cluster gli (14), now known to contain 13 genes, the function of a number of cluster genes has been established by a combination of targeted gene deletion and phenotypic and biochemical analyses (4, 10, 11, 13, 22, 31-33, 34, 36).…”