RESUMO
EmrE is a small multidrug resistance transporter found in Escherichia coli that confers resistance to toxic polyaromatic cations due to its proton-coupled antiport of these substrates. Here we show that EmrE breaks the rules generally deemed essential for coupled antiport. NMR spectra reveal that EmrE can simultaneously bind and cotransport proton and drug. The functional consequence of this finding is an exceptionally promiscuous transporter: not only can EmrE export diverse drug substrates, it can couple antiport of a drug to either one or two protons, performing both electrogenic and electroneutral transport of a single substrate. We present a free-exchange model for EmrE antiport that is consistent with these results and recapitulates ∆pH-driven concentrative drug uptake. Kinetic modeling suggests that free exchange by EmrE sacrifices coupling efficiency but boosts initial transport speed and drug release rate, which may facilitate efficient multidrug efflux.
Assuntos
Antiporters/química , Farmacorresistência Bacteriana Múltipla/genética , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Oniocompostos/metabolismo , Compostos Organofosforados/metabolismo , Prótons , Xenobióticos/metabolismo , Antiporters/genética , Antiporters/metabolismo , Sítios de Ligação , Transporte Biológico , Dicicloexilcarbodi-Imida/toxicidade , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Cinética , Simulação de Dinâmica Molecular , Oniocompostos/química , Oniocompostos/farmacologia , Compostos Organofosforados/química , Compostos Organofosforados/farmacologia , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilgliceróis/química , Fosfatidilgliceróis/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteolipídeos/química , Proteolipídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica , Xenobióticos/química , Xenobióticos/farmacologiaRESUMO
The continued increase in the size of the protein sequence databases as a result of advances in genome sequencing technology is overwhelming the ability to perform experimental characterization of function. Consequently, functions are assigned to the vast majority of proteins via automated, homology-based methods, with the result that as many as 50% are incorrectly annotated or unannotated ( Schnoes et al. PLoS Comput. Biol. 2009 , 5 ( 12 ), e1000605 ). This manuscript describes a study of the D-mannonate dehydratase (ManD) subgroup of the enolase superfamily (ENS) to investigate how function diverges as sequence diverges. Previously, one member of the subgroup had been experimentally characterized as ManD [dehydration of D-mannonate to 2-keto-3-deoxy-D-mannonate (equivalently, 2-keto-3-deoxy-D-gluconate)]. In this study, 42 additional members were characterized to sample sequence-function space in the ManD subgroup. These were found to differ in both catalytic efficiency and substrate specificity: (1) high efficiency (kcat/KM = 10(3) to 10(4) M(-1) s(-1)) for dehydration of D-mannonate, (2) low efficiency (kcat/KM = 10(1) to 10(2) M(-1) s(-1)) for dehydration of d-mannonate and/or D-gluconate, and 3) no-activity with either D-mannonate or D-gluconate (or any other acid sugar tested). Thus, the ManD subgroup is not isofunctional and includes D-gluconate dehydratases (GlcDs) that are divergent from the GlcDs that have been characterized in the mandelate racemase subgroup of the ENS (Lamble et al. FEBS Lett. 2004 , 576 , 133 - 136 ) (Ahmed et al. Biochem. J. 2005 , 390 , 529 - 540 ). These observations signal caution for functional assignment based on sequence homology and lay the foundation for the studies of the physiological functions of the GlcDs and the promiscuous ManDs/GlcDs.