The genes lmbA,B1,B2 in the lincomycin A production gene cluster of Streptomyces lincolnensis were shown to form a common transcription unit with the promoter located directly upstream of lmbA. The proteins LmbB1 (mol. mass, 18 kDa) and LmbB2 (mol. mass 34 kDa), when over-produced together in Escherichia coli, brought about enzyme activities for the specific conversion of both L-tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA) to a yellow-colored product. The LmbB1 protein alone catalyzed the conversion of L-DOPA, but not of L-tyrosine. The purified LmbB1 protein showed a Km for L-DOPA of 258.3 microM. The L-tyrosine converting activity could not been demonstrated in vitro. The preliminary interpretation of these data suggests that the protein LmbB1 is an L-DOPA extradiol-cleaving 2,3-dioxygenase and that the protein LmbB2, either alone or in accord with LmbB1, represents an L-tyrosine 3-hydroxylase. This sequence of putative oxidation reactions on L-tyrosine seems to represent a new pathway different from the ones catalyzed by mammalian L-tyrosine hydroxylases or the wide-spread tyrosinases. The protein LmbA seemed not to be involved in this process. The labile, yellow-colored product from L-DOPA could not be converted to a picolinic acid derivative [3-(2-carboxy-5-pyridyl)alanine] in the presence of ammonia. Therefore, it probably is not a derivative of a cis, cis-3-hydroxymuconic acid semialdehyde; instead, its speculative structure represents a heterocyclic precursor of the propylhygric acid moiety of lincomycin A.

The gene pkwA coding for a typical WD-repeat protein was found in the chromosome of the bacterium Thermomonospora curvata CCM 3352. Until now WD-repeat proteins were through to be confined to eukaryotes.

The gene lmbB2 of the lincomycin biosynthetic gene cluster of Streptomyces lincolnensis ATCC 25466 was shown to code for an unusual tyrosine hydroxylating enzyme involved in the biosynthetic pathway of this clinically important antibiotic. LmbB2 was expressed in Escherichia coli, purified near to homogeneity and shown to convert tyrosine to 3,4-dihydroxyphenylalanine (DOPA). In contrast to the well-known tyrosine hydroxylases (EC 1.14.16.2) and tyrosinases (EC 1.14.18.1), LmbB2 was identified as a heme protein. Mass spectrometry and Soret band-excited Raman spectroscopy of LmbB2 showed that LmbB2 contains heme b as prosthetic group. The CO-reduced differential absorption spectra of LmbB2 showed that the coordination of Fe was different from that of cytochrome P450 enzymes. LmbB2 exhibits sequence similarity to Orf13 of the anthramycin biosynthetic gene cluster, which has recently been classified as a heme peroxidase. Tyrosine hydroxylating activity of LmbB2 yielding DOPA in the presence of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) was also observed. Reaction mechanism of this unique heme peroxidases family is discussed. Also, tyrosine hydroxylation was confirmed as the first step of the amino acid branch of the lincomycin biosynthesis.