RESUMO
Bacterial coenzyme B12-dependent 2-hydroxyisobutyryl-CoA mutase (HCM) is a radical enzyme catalyzing the stereospecific interconversion of (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA. It consists of two subunits, HcmA and HcmB. To characterize the determinants of substrate specificity, we have analyzed the crystal structure of HCM from Aquincola tertiaricarbonis in complex with coenzyme B12 and the substrates (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in alternative binding. When compared with the well studied structure of bacterial and mitochondrial B12-dependent methylmalonyl-CoA mutase (MCM), HCM has a highly conserved domain architecture. However, inspection of the substrate binding site identified amino acid residues not present in MCM, namely HcmA Ile(A90) and Asp(A117). Asp(A117) determines the orientation of the hydroxyl group of the acyl-CoA esters by H-bond formation, thus determining stereospecificity of catalysis. Accordingly, HcmA D117A and D117V mutations resulted in significantly increased activity toward (R)-3-hydroxybutyryl-CoA. Besides interconversion of hydroxylated acyl-CoA esters, wild-type HCM as well as HcmA I90V and I90A mutant enzymes could also isomerize pivalyl- and isovaleryl-CoA, albeit at >10 times lower rates than the favorite substrate (S)-3-hydroxybutyryl-CoA. The nonconservative mutation HcmA D117V, however, resulted in an enzyme showing high activity toward pivalyl-CoA. Structural requirements for binding and isomerization of highly branched acyl-CoA substrates such as 2-hydroxyisobutyryl- and pivalyl-CoA, possessing tertiary and quaternary carbon atoms, respectively, are discussed.
Assuntos
Acil Coenzima A/metabolismo , Proteínas de Bactérias/metabolismo , Cobamidas/metabolismo , Hidroxibutiratos/metabolismo , Transferases Intramoleculares/metabolismo , Acil Coenzima A/química , Acil Coenzima A/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Betaproteobacteria/enzimologia , Betaproteobacteria/genética , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Transferases Intramoleculares/química , Transferases Intramoleculares/genética , Cinética , Metilmalonil-CoA Mutase/química , Metilmalonil-CoA Mutase/genética , Metilmalonil-CoA Mutase/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Homologia de Sequência de Aminoácidos , Estereoisomerismo , Especificidade por SubstratoRESUMO
Aerobic anoxygenic photosynthesis (AAP) is found in an increasing number of proteobacterial strains thriving in ecosystems ranging from extremely oligotrophic to eutrophic. Here, we have investigated whether the fuel oxygenate-degrading betaproteobacterium Aquincola tertiaricarbonis L108 can use AAP to compensate kinetic limitations at low heterotrophic substrate fluxes. In a fermenter experiment with complete biomass retention and also during chemostat cultivation, strain L108 was challenged with extremely low substrate feeding rates of tert-butyl alcohol (TBA), an intermediate of methyl tert-butyl ether (MTBE). Interestingly, formation of photosynthetic pigments, identified as bacteriochlorophyll a and spirilloxanthin, was only induced in growing cells at TBA feeding rates less than or equal to maintenance requirements observed under energy excess conditions. Growth continued at rates between 0.001 and 0.002 h(-1) even when the TBA feed was decreased to values close to 30â% of this maintenance rate. Partial sequencing of genomic DNA of strain L108 revealed a bacteriochlorophyll synthesis gene cluster (bchFNBHL) and photosynthesis regulator genes (ppsR and ppaA) typically found in AAP and other photosynthetic proteobacteria. The usage of light as auxiliary energy source enabling evolution of efficient degradation pathways for kinetically limited heterotrophic substrates and for lowering the threshold substrate concentration Smin at which growth becomes zero is discussed.
