Several Sphingomonas spp. utilize polyethylene glycols (PEGs) as a sole carbon and energy source, oxidative PEG degradation being initiated by a dye-linked dehydrogenase (PEG-DH) that oxidizes the terminal alcohol groups of the polymer chain. Purification and characterization of PEG-DH from Sphingomonas terrae revealed that the enzyme is membrane bound. The gene encoding this enzyme (pegA) was cloned, sequenced, and expressed in Escherichia coli. The purified recombinant enzyme was vulnerable to aggregation and inactivation, but this could be prevented by addition of detergent. It is as a homodimeric protein with a subunit molecular mass of 58.8 kDa, each subunit containing 1 noncovalently bound flavin adenine dinucleotide but not Fe or Zn. PEG-DH recognizes a broad variety of primary aliphatic and aromatic alcohols as substrates. Comparison with known sequences revealed that PEG-DH belongs to the group of glucose-methanol-choline (GMC) flavoprotein oxidoreductases and that it is a novel type of flavoprotein alcohol dehydrogenase related (percent identical amino acids) to other, so far uncharacterized bacterial, membrane-bound, dye-linked dehydrogenases: alcohol dehydrogenase from Pseudomonas oleovorans (46%); choline dehydrogenase from E. coli (40%); L-sorbose dehydrogenase from Gluconobacter oxydans (38%); and 4-nitrobenzyl alcohol dehydrogenase from a Pseudomonas species (35%).Polyethylene glycols (PEGs) are industrially produced in large quantities and in a broad spectrum of sizes and derivatives. In view of the application of these xenobiotics, after use they mainly show up in sewage water but are easily biologically degraded by many aerobic bacteria belonging to different genera (3,19).PEG metabolization has been reported for several sphingomonads, either in an axenic (e.g., S. macrogoltabidus [8]) or in a mixed culture (e.g., S. terrae with a Rhizobium sp. [4]). In all cases, it has been established that the first step in the degradation pathway involves the oxidation of the terminal alcohol groups with a dye-linked dehydrogenase (5), here called PEG-DH. Originally the enzyme was considered to be a quinoprotein (7), as in Rhodopseudomonas acidophila (20,21), but this could not be proven because the difficulties encountered in the purification of this membrane-bound enzyme prevented straightforward characterization and cofactor identification of it. However, sufficiently pure enzyme could be prepared for N-terminal amino acid sequence determination. Cloning of the gene (pegA) and overexpression of it in an Escherichia coli recombinant strain provided the quantities of enzyme required for full characterization. It is shown here that PEG-DH is not a quinoprotein but a new type of flavoprotein dehydrogenase with flavin adenine dinucleotide (FAD) as a cofactor.Purification of PEG-DH from S. terrae and sequencing of its N-terminal amino acids. PEG-DH from S. terrae was purified from a symbiotic mixed culture (E-1) consisting of S. terrae and a Rhizobium sp. Growth and purification were carried out essentially a...

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