Determination of the metal ion dependence and substrate specificity of a hydratase involved in the degradation pathway of biphenyl/chlorobiphenyl.
BphH is a divalent metal ion-dependent hydratase that catalyzes the formation of 2-keto-4-hydroxypentanoate from 2-hydroxypent-2,4-dienoate (HPDA). This reaction lies on the catabolic pathway of numerous aromatics, including the significant environmental pollutant, polychlorinated biphenyls (PCBs). BphH from the PCB degrading bacterium, Burkholderia xenoverans LB400, was overexpressed and purified to homogeneity. Atomic absorption spectroscopy and Scatchard analysis reveal that only one divalent metal ion is bound to each enzyme subunit. The enzyme exhibits the highest activity when Mg2+ was used as cofactor. Other divalent cations activate the enzyme in the following order of effectiveness: Mg2+ > Mn2+ > Co2+ > Zn2+ > Ca2+. This differs from the metal activation profile of the homologous hydratase, MhpD. UV-visible spectroscopy of the Co2+-BphH complex indicates that the divalent metal ion is hexa-coordinated in the enzyme. The nature of the metal ion affected only the kcat and not the Km values in the BphH hydration of HPDA, suggesting that cation has a catalytic rather than just a substrate binding role. BphH is able to transform alternative substrates substituted with methyl- and chlorine groups at the 5-position of HPDA. The specificity constants (kcat/Km) for 5-methyl and 5-chloro substrates are, however, lowered by eight- and 67-fold compared with the unsubstituted substrate. Significantly, kcat for the chloro-substituted substrate is eightfold lower compared with the methyl-substituted substrate, showing that electron withdrawing substituent at the 5-position of the substrate has a negative influence on enzyme catalysis.
Wang
Pan
P
Department of Microbiology, University of Guelph, Ontario, Canada.
Seah
Stephen Y K
SY
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001713:Biphenyl Compounds / Q000378:metabolism*; D019117:Burkholderia / Q000201:enzymology / Q000235:genetics; D002412:Cations / Q000378:metabolism; D003001:Cloning, Molecular; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism*; D006843:Hydrocarbons, Chlorinated / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000378:metabolism*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D001713:Biphenyl Compounds; D002412:Cations; D005231:Fatty Acids, Unsaturated; D006836:Hydro-Lyases; D006843:Hydrocarbons, Chlorinated; D008670:Metals; C010574:biphenyl; C061660:2-hydroxy-2,4-pentadienoic acid