l‐3,4‐Dihydroxyphenyl alanine‐extradiol cleavage is followed by intramolecular cyclization in lincomycin biosynthesis

Abstract

The LmbB1 protein, participating in the biosynthesis of lincomycin, was heterologously expressed in Escherichia coli, purified in its active form, and characterized as a dimer of identical subunits. Methods for purification and analysis of the LmbB1 reaction product were developed. Molecular mass and fragmentation pattern of the product revealed by capillary electrophoresis-mass spectrometry were in agreement with its proposed structure, 4-(3-carboxy-3-oxopropenyl)-2,3-dihydro-1H-pyrrole-2-carboxylic acid. The LmbB1 is therefore a dioxygenase catalysing the 2,3-extradiol cleavage of the L-3,4-dihydroxyphenyl alanine aromatic ring. The final LmbB1 reaction product, a unique compound found in biosynthesis of lincomycin and expected in anthramycins, arises through subsequent cyclization of the primary cleavage product, 2,3-secodopa. A possible role of LmbB1 in 2,3-secodopa cyclization and alternative ways of the cyclization in the formation of biosynthetically related compounds, muscaflavin and stizolobinic acid, are discussed.Keywords: lincomycin; L-DOPA; derivative of muconic acid semialdehyde; 2,3-extradiol dioxygenase; 4-(3-carboxy-3-oxo-propenyl)-2,3-dihydro-1H-pyrrole-2-carboxylic acid.Lincomycin A and, particularly, its semisynthetic derivative clindamycin belong to clinically important antibiotics, yet, our knowledge of lincomycin biosynthesis is rather fragmentary.Structurally, lincomycin A is composed of two moieties, a sugar moiety 6-amino-6,8-dideoxy-1-thio-D-erythroa-D-galactooctopyranoside (methylthiolincosaminide) and an amino acid derivative 4-n-propyl-L-hygric acid, linked by an amide bond. A pathway leading to 4-n-propyl-L-hygric acid moiety formation was proposed based on determination of the carbon and nitrogen atoms biosynthetic origin using feeding experiments with subsequent NMR analysis [1]. The 4-n-propyl-L-hygric acid moiety is derived from L-tyrosine, which is hydroxylated to yield L-3,4-dihydroxyphenyl alanine (L-DOPA). The subsequent steps include 2,3-extradiol cleavage of the L-DOPA aromatic ring, cyclization to produce the five-membered ring containing nitrogen (Fig. 1), loss of two carbons, C-methylation at the aliphatic side chain and double reduction to yield propylproline. After condensation of the sugar and aglycon moieties into N-demethyllincomycin, the aglycon is N-methylated to yield the 4-n-propyl-L-hygric acid moiety. Although the scheme of the pathway was drawn in 1984, the one and only intermediate of the multistep L-DOPA conversion to propylproline, 1,2,3,6-tetradehydro-propylproline, was identified [2].Sequence analysis of the Streptomyces lincolnensis lincomycin biosynthetic gene cluster revealed 29 ORFs with putative biosynthetic or regulatory (lmb genes) and resistance functions (lmr genes) [3,4]. Assigning functions to the proteins encoded by the lmb genes is considerably complicated by limited information on the intermediates of the lincomycin biosynthetic pathway. The function has been demonstrated experimentally for three of the lmb genes: lmbJ [5], which codes fo...

The Novel Transcriptional Regulator LmbU Promotes Lincomycin Biosynthesis through Regulating Expression of Its Target Genes in Streptomyces lincolnensis

“…Protein sequence alignments show that LmbU from S. lincolnensis shares 48% to 72% identity with a number of homologues from other actinomycetes (see Table S1 in the supplemental material), some of which are located in biosynthetic gene clusters of secondary metabolites, such as HrmB (GenBank accession no. AEH41782.1) in the hormaomycin gene cluster from Streptomyces griseoflavus (12), HmtD (GenBank accession no. CBZ42138.1) in the himastatin gene cluster from Streptomyces himastatinicus, and AcmO (GenBank accession no.…”

“…L-Tyrosine was identified as a precursor of PPL through stable-isotope labeling (10), and it is converted into L-3,4-dihydroxyphenylalanine (L-DOPA) and then into 4-(3-carboxy-3-oxo-propenyl)-2,3-dihydro-1H-pyrrole-2-carboxylic acid (product 1) by the L-tyrosine hydroxylase LmbB2 and the L-DOPA-2,3-dioxygenase LmbB1 (11,12). In the recent report, it was proposed that product 1 undergoes methylation by LmbW to generate product 2 (13,14).…”

Biosynthesis of Sibiromycin, a Potent Antitumor Antibiotic

“…Labeling experiments have identified L-tyrosine as the biosynthetic precursor for the hydropyrrole moieties of PBDs, as well as of lincomycin A (2, 11). The functional assignment of the biosynthetic enzymes involved in this transformation with the exception of the first two steps catalyzed by a tyrosine hydroxylase and an L-DOPA 2,3-dioxygenase (31,33) remains undetermined in the PBDs and lincomycin A biosyntheses (10,37). We propose a common biosynthetic route for the formation of 4-propylidene-tetrahydropyrrole-2-carboxylic acid in the PBDs and lincomycin biosyntheses, the last intermediate in common in the anthramycin, sibiromycin, and lincomycin A biosyntheses (Fig.…”

Cinnamic Acid, an Autoinducer of Its Own Biosynthesis, Is Processed via Hca Enzymes in Photorhabdus luminescens

“…The Hca dioxygenase is involved in this degradation. The first steps of lincomycin A biosynthesis in Streptomyces lincolnensis comprise the oxidation of the aromatic ring of tyrosine by dioxygenases (28). Thus, the Hca dioxygenase could catalyze the first step of the ST biosynthetic pathway in P. luminescens.…”