Abstract
Grixazone contains a phenoxazinone chromophore and is a secondary metabolite produced by Streptomyces griseus. In the grixazone biosynthesis gene cluster, griF (encoding a tyrosinase homolog) and griE (encoding a protein similar to copper chaperons for tyrosinases) are encoded. An expression study of GriE and GriF in Escherichia coli showed that GriE activated GriF by transferring copper ions to GriF, as has been observed for a Streptomyces melanogenesis system in which the MelC1 copper chaperon transfers copper ions to MelC2 tyrosinase. In contrast with tyrosinases, GriF showed no monophenolase activity, although it oxidized various o-aminophenols as preferable substrates rather than catechol-type substrates. Deletion of the griEF locus on the chromosome resulted in accumulation of 3-amino-4-hydroxybenzaldehyde (3,4-AHBAL) and its acetylated compound, 3-acetylamino-4-hydroxybenzaldehyde. GriF oxidized 3,4-AHBAL to yield an o-quinone imine derivative, which was then non-enzymatically coupled with another molecule of the o-quinone imine to form a phenoxazinone. The coexistence of N-acetylcysteine in the in vitro oxidation of 3,4-AH-BAL by GriF resulted in the formation of grixazone A, suggesting that the -SH group of N-acetylcysteine is conjugated to the o-quinone imine formed from 3,4-AHBAL and that the conjugate is presumably coupled with another molecule of the o-quinone imine. GriF is thus a novel o-aminophenol oxidase that is responsible for the formation of the phenoxazinone chromophore in the grixazone biosynthetic pathway.
Highlights
We determined the plausible substrate of GriF by structural elucidation of an intermediate (3-amino-4-hydroxybenzaldehyde (3,4-AHBAL3; compound 2a) that accumulated in a griEFdeleted mutant and revealed the in vitro formation of a phenoxazinone skeleton from 3,4-AHBAL by recombinant GriF produced in Escherichia coli
Functions of GriF and GriE—GriF encoded within the grixazone biosynthesis gene cluster has been found to be responsible for the formation of the phenoxazinone chromophore of grixazone by oxidative coupling of o-aminophenols
Streptomycetes produce a variety of secondary metabolites with a phenoxazinone chromophore, and some of the tyrosinase homologs are supposed to be responsible for the formation of phenoxazinones
Summary
E. coli strains JM109 and TOP10 (Invitrogen) and plasmids pUC19 and pCR4Blunt-TOPO (Invitrogen), pET17b, and pET-26b were used for DNA manipulation. A 3.6-kb NcoI-SphI fragment containing a region upstream from griE and a 3.6-kb PmaCI fragment containing a 3Ј-portion of griF and a region downstream from griF were connected with a short linker derived from the multi-cloning linker of pUC19. This 7.2-kb fragment, which had a 1.1-kb deletion of the sequence in the griEF locus (from Met of GriE to His226 of GriF), was cloned into pUC19 together with a 1.1-kb HindIII fragment carrying the kanamycin resistance gene from Tn5, resulting in p⌬griEF.
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