RESUMO
cis-Prenyltransferases (cis-PTs) catalyze consecutive condensations of isopentenyl diphosphate to an allylic diphosphate acceptor to produce a linear polyprenyl diphosphate of designated length. Dimer formation is a prerequisite for cis-PTs to catalyze all cis-prenyl condensation reactions. The structure-function relationship of a conserved C-terminal RXG motif in cis-PTs that forms inter-subunit interactions and has a role in catalytic activity has attracted much attention. Here, we solved the crystal structure of a medium-chain cis-PT from Thermobifida fusca that produces dodecaprenyl diphosphate as a polyprenoid glycan carrier for cell wall synthesis. The structure revealed a characteristic dimeric architecture of cis-PTs in which a rigidified RXG motif of one monomer formed inter-subunit hydrogen bonds with the catalytic site of the other monomer, while the RXG motif of the latter remained flexible. Careful analyses suggested the existence of a possible long-range negative cooperativity between the two catalytic sites on the two monomeric subunits that allowed the binding of one subunit to stabilize the formation of the enzyme-substrate ternary complex and facilitated the release of Mg-PPi and subsequent intra-molecular translocation at the counter subunit so that the condensation reaction could occur in consecutive cycles. The current structure reveals the dynamic nature of the RXG motif and provides a rationale for pursuing further investigations to elucidate the inter-subunit cooperativity of cis-PTs.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Transferases/química , Transferases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Biocatálise , Domínio Catalítico , Sequência Conservada , Cristalografia por Raios X , Estabilidade Enzimática , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas , Thermobifida/enzimologia , Thermobifida/genética , Transferases/genéticaRESUMO
cis-Prenyltransferase catalyzes the synthesis of Z,E-mixed prenyl diphosphates by sequential condensation of isopentenyl diphosphate with allylic diphosphate. cis-Prenyltransferases can be classified into three subgroups: short-, medium-, and long-chain cis-prenyltransferase, according to their product chain lengths. cis-Farnesyl diphosphate synthase from Mycobacterium tuberculosis has been the only example as short-chain cis-prenyltransferase so far characterized. In this study, we cloned the novel short-chain cis-prenyltransferases from three different bacteria, and characterized their enzymatic activities to compare and elucidate a common feature of the short-chain cis-prenyltransferases. Furthermore, we identified a specific isoleucine that is conserved in short-chain cis-prenyltransferases and located in close proximity of the omega-end of the geranyl diphosphate. Several site-directed mutants with respect to the isoleucine residue synthesized longer prenyl chain products and showed broader allylic substrate specificity. These results suggested that the isoleucine plays an important role in the substrate specificity and chain length determination mechanism of cis-prenyltransferase.
Assuntos
Actinomycetales/enzimologia , Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/enzimologia , Transferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , Clonagem Molecular , Isoleucina/genética , Isoleucina/metabolismo , Mutagênese Sítio-Dirigida , Alinhamento de Sequência , Especificidade por Substrato , Transferases/química , Transferases/genéticaRESUMO
cis-Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic prenyl diphosphates, producing Z,E-mixed prenyl diphosphate. The Mycobacterium tuberculosis Z,E-farnesyl diphosphate synthase Rv1086 catalyzes the condensation of one molecule of IPP with geranyl diphosphate to yield Z,E-farnesyl diphosphate and is classified as a short-chain cis-prenyltransferase. To elucidate the chain-length determination mechanism of the short-chain cis-prenyltransferase, we introduced some substitutive mutations at the characteristic amino acid residues of Rv1086. Among the mutants constructed, L84A showed a dramatic change of catalytic function to synthesize longer prenyl chain products than that of wild type, indicating that Leu84 of Rv1086 plays an important role in product chain-length determination. Mutagenesis at the corresponding residue of a medium-chain cis-prenyltransferase, Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase also resulted in the production of different prenyl chain length from the intrinsic product, suggesting that this position also plays an important role in product chain-length determination for medium-chain cis-prenyltransferases.
Assuntos
Alquil e Aril Transferases/química , Proteínas de Bactérias/química , Geraniltranstransferase/química , Leucina/química , Mycobacterium tuberculosis/enzimologia , Alquil e Aril Transferases/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Catálise , Sequência Conservada , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/genética , Geraniltranstransferase/genética , Leucina/genética , Micrococcus luteus/enzimologia , Micrococcus luteus/genética , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Estrutura Secundária de Proteína/genética , Alinhamento de SequênciaRESUMO
cis-Prenyltransferase catalyzes the synthesis of Z,E-mixed prenyl diphosphates by a condensation of isopentenyl diphosphate to an allylic diphosphate. A novel gene encoding a cis-prenyltransferase is cloned from Thermobifida fusca. It showed a unique substrate specificity accepting dimethylallyl diphosphate as a shortest allylic substrate, and synthesizes polyprenyl products up to C(70).