Lincomycin biosynthesis involves a tyrosine hydroxylating heme protein of an unusual enzyme family

PLoS One. 2013 Dec 4;8(12):e79974. doi: 10.1371/journal.pone.0079974. eCollection 2013.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / biosynthesis*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Chromatography, High Pressure Liquid
  • Circular Dichroism
  • Dihydroxyphenylalanine / metabolism
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Gene Expression
  • Heme / chemistry
  • Heme / metabolism
  • Hemeproteins / genetics
  • Hemeproteins / metabolism*
  • Hydroxylation
  • Iron / chemistry
  • Iron / metabolism
  • Lincomycin / biosynthesis*
  • Multigene Family
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Streptomyces / enzymology*
  • Streptomyces / genetics
  • Tyrosine / metabolism
  • Tyrosine 3-Monooxygenase / genetics
  • Tyrosine 3-Monooxygenase / metabolism*

Substances

  • Anti-Bacterial Agents
  • Bacterial Proteins
  • Hemeproteins
  • Recombinant Proteins
  • Tyrosine
  • Heme
  • Dihydroxyphenylalanine
  • Lincomycin
  • Iron
  • Tyrosine 3-Monooxygenase

Grants and funding

This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (CZ.1.07/2.3.00/20.0055, CZ.1.07/2.3.00/30.0003, MSM0021620835, MSM0021622412, and LC06010) URL: http://www.msmt.cz/index.php?lang=2; the Grant Agency of the Charles University in Prague (204/2006/B-FYZ/MFF) URL: http://www.cuni.cz/UKENG-33.html; Grant Agency of the Academy of Sciences of the Czech Republic (IAA500200810) URL: http://www.gaav.cz/index2.html; Ministry of Agriculture of the Czech Republic, National Agency for Agriculture Research (QH92151) URL: http://www.ldf.mendelu.cz/en/projekty/resene_projekty/grantove_prilezitosti/nazv; Slovak Grant Agency (Grant VEGA 2/0122/11), the European Regional Development fund (CZ.1.05/1.1.00/02.0068 (CEITEC)) and Institutional Research Concept AV OZ 50200510 URL: http://www.biomed.cas.cz/mbu/new/index.php?p=projekty&site=en. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.