Quinohaemoprotein ethanol dehydrogenase from Comamonas testosteroni. Purification, characterization, and reconstitution of the apoenzyme with pyrroloquinoline quinone analogues.
Pyrroloquinoline-quinone(PQQ)-free quinohaemoprotein ethanol dehydrogenase (QH-EDH) apoenzyme was isolated from ethanol-grown Comamonas testosteroni. The purified apoenzyme, showing a single band of 71 kDa on native gel electrophoresis, could be only partially converted into active holoenzyme by addition of PQQ in the presence of calcium ions. In addition to a band with a molecular mass of 71 kDa, additional bands of 51 kDa and 25 kDa were observed with SDS/PAGE. Analysis of the N-terminal sequences of the bands and comparison with the DNA sequence of the gene, suggested that the latter two originate from the former one, due to scission occurring at a specific site between two vicinal residues in the protein chain. The extent of scission appeared to increase during growth of the organism. After addition of PQQ to apoenzyme, holoenzyme and nicked, inactive enzyme could be separated. Holoenzyme prepared in this way was found to contain equimolar amounts of PQQ, Ca2+ and covalently bound haem. EPR spectra of fully oxidized apo-QH-EDH and holo-QH-EDH showed g values typical for low-spin haem c proteins. In partially oxidized holo-QH-EDH an organic radical signal attributed to the semiquinone form of PQQ was observed. Binding of PQQ leads to conformational changes, as reflected by changes of spectral and chromatographic properties. Reconstitution of apoenzyme with PQQ analogues resulted in a decreased activity and enantioselectivity for the oxidation of chiral alcohols. Compared with PQQ, analogues with a large substituent had a lower affinity for the apoenzyme. Results with other analogues indicated that possession of the o-quinone/o-quinol moiety is not essential for binding but it is for activity.
de Jong
G A
GA
Department of Microbiology and Enzymology, Delft University of Technology, The Netherlands.
Geerlof
A
A
Stoorvogel
J
J
Jongejan
J A
JA
de Vries
S
S
Duine
J A
JA
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D008969:Molecular Sequence Data; D045542:PQQ Cofactor; D015363:Quinolones / Q000494:pharmacology*; D013056:Spectrophotometry, Ultraviolet
D001051:Apoenzymes; D015363:Quinolones; D045542:PQQ Cofactor; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor)