QUINOHEMOPROTEIN ETHANOL DEHYDROGENASE FROM COMAMONAS-TESTOSTERONI - PURIFICATION, CHARACTERIZATION, AND RECONSTITUTION OF THE APOENZYME WITH PYRROLOQUINOLINE QUINONE ANALOGS

Pyrroloquinoline-quinone(PQQ)-free quinohaemoprotein ethanol dehydroge nase (QH-EDH) apoenzyme was isolated from ethanol-grown Comamonas test osteroni. The purified apoenzyme, showing a single band of 71 kDa on n ative gel electrophoresis, could be only partially converted into acti ve holoenzyme by addition of PQQ in the presence of calcium ions. In a ddition 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-term inal sequences of the bands and comparison with the DNA sequence of th e gene, suggested that the latter two originate from the former one, d ue to scission occurring at a specific site between two vicinal residu es in the protein chain. The extent of scission appeared to increase d uring growth of the organism. After addition of PQQ to apoenzyme, holo enzyme and nicked, inactive enzyme could he separated. Holoenzyme prep ared in this way was found to contain equimolar amounts of PQQ, Ca2+ a nd 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 con formational changes, as reflected by changes of spectral and chromatog raphic properties. Reconstitution of apoenzyme with PQQ analogues resu lted in a decreased activity and enantioselectivity for the oxidation of chiral alcohols. Compared with PQQ, analogues with a large substitu ent had a lower affinity for the apoenzyme. Results with ether analogu es indicated that possession of the o-quinone/o-quinol moiety is not e ssential for binding but it is for activity.