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The yeast mitochondrial citrate transport protein: molecular determinants of its substrate specificity.

ABSTRACT: The objective of this study was to identify the role of individual amino acid residues in determining the substrate specificity of the yeast mitochondrial citrate transport protein (CTP). Previously, we showed that the CTP contains at least two substrate-binding sites. In this study, utilizing the overexpressed, single-Cys CTP-binding site variants that were functionally reconstituted in liposomes, we examined CTP specificity from both its external and internal surfaces. Upon mutation of residues comprising the more external site, the CTP becomes less selective for citrate with numerous external anions able to effectively inhibit [(14)C]citrate/citrate exchange. Thus, the site 1 variants assume the binding characteristics of a nonspecific anion carrier. Comparison of [(14)C]citrate uptake in the presence of various internal anions versus water revealed that, with the exception of the R189C mutant, the other site 1 variants showed substantial uniport activity relative to exchange. Upon mutation of residues comprising site 2, we observed two types of effects. The K37C mutant displayed a markedly enhanced selectivity for external citrate. In contrast, the other site 2 mutants displayed varying degrees of relaxed selectivity for external citrate. Examination of internal substrates revealed that, in contrast to the control transporter, the R181C variant exclusively functioned as a uniporter. This study provides the first functional information on the role of specific binding site residues in determining mitochondrial transporter substrate selectivity. We interpret our findings in the context of our homology-modeled CTP as it cycles between the outward-facing, occluded, and inward-facing states.

SUBMITTER: Aluvila S

PROVIDER: S-EPMC2930730 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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The yeast mitochondrial citrate transport protein: molecular determinants of its substrate specificity.

Aluvila Sreevidya S Kotaria Rusudan R Sun Jiakang J Mayor June A JA Walters D Eric DE Harrison David H T DHT Kaplan Ronald S RS

The Journal of biological chemistry 20100615 35

The objective of this study was to identify the role of individual amino acid residues in determining the substrate specificity of the yeast mitochondrial citrate transport protein (CTP). Previously, we showed that the CTP contains at least two substrate-binding sites. In this study, utilizing the overexpressed, single-Cys CTP-binding site variants that were functionally reconstituted in liposomes, we examined CTP specificity from both its external and internal surfaces. Upon mutation of residue ...[more]

PMID: 20551333

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Project description:5'-Methylthioadenosine (MTA)/S-adenosylhomocysteine (SAH) nucleosidase (MTAN) is essential for cellular metabolism and development in many bacterial species. While the enzyme is found in plants, plant MTANs appear to select for MTA preferentially, with little or no affinity for SAH. To understand what determines substrate specificity in this enzyme, MTAN homologues from Arabidopsis thaliana (AtMTAN1 and AtMTAN2, which are referred to as AtMTN1 and AtMTN2 in the plant literature) have been characterized kinetically. While both homologues hydrolyze MTA with comparable kinetic parameters, only AtMTAN2 shows activity towards SAH. AtMTAN2 also has higher catalytic activity towards other substrate analogues with longer 5'-substituents. The structures of apo AtMTAN1 and its complexes with the substrate- and transition-state-analogues, 5'-methylthiotubercidin and formycin A, respectively, have been determined at 2.0-1.8 A resolution. A homology model of AtMTAN2 was generated using the AtMTAN1 structures. Comparison of the AtMTAN1 and AtMTAN2 structures reveals that only three residues in the active site differ between the two enzymes. Our analysis suggests that two of these residues, Leu181/Met168 and Phe148/Leu135 in AtMTAN1/AtMTAN2, likely account for the divergence in specificity of the enzymes. Comparison of the AtMTAN1 and available Escherichia coli MTAN (EcMTAN) structures suggests that a combination of differences in the 5'-alkylthio binding region and reduced conformational flexibility in the AtMTAN1 active site likely contribute to its reduced efficiency in binding substrate analogues with longer 5'-substituents. In addition, in contrast to EcMTAN, the active site of AtMTAN1 remains solvated in its ligand-bound forms. As the apparent pK(a) of an amino acid depends on its local environment, the putative catalytic acid Asp225 in AtMTAN1 may not be protonated at physiological pH and this suggests the transition state of AtMTAN1, like human MTA phosphorylase and Streptococcus pneumoniae MTAN, may be different from that found in EcMTAN.

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The yeast mitochondrial citrate transport protein: molecular determinants of its substrate specificity.

Publications

The yeast mitochondrial citrate transport protein: molecular determinants of its substrate specificity.

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