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Structural basis for the aldolase and epimerase activities of Staphylococcus aureus dihydroneopterin aldolase.

ABSTRACT: Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) and the epimerization of DHNP to 7,8-dihydromonopterin (DHMP). Although crystal structures of the enzyme from several microorganisms have been reported, no structural information is available about the critical interactions between DHNA and the trihydroxypropyl moiety of the substrate, which undergoes bond cleavage and formation. Here, we present the structures of Staphylococcus aureus DHNA (SaDHNA) in complex with neopterin (NP, an analog of DHNP) and with monapterin (MP, an analog of DHMP), filling the gap in the structural analysis of the enzyme. In combination with previously reported SaDHNA structures in its ligand-free form (PDB entry 1DHN) and in complex with HP (PDB entry 2DHN), four snapshots for the catalytic center assembly along the reaction pathway can be derived, advancing our knowledge about the molecular mechanism of SaDHNA-catalyzed reactions. An additional step appears to be necessary for the epimerization of DHMP to DHNP. Three active site residues (E22, K100, and Y54) function coordinately during catalysis: together, they organize the catalytic center assembly, and individually, each plays a central role at different stages of the catalytic cycle.

SUBMITTER: Blaszczyk J

PROVIDER: S-EPMC1885205 | biostudies-literature | 2007 Apr

REPOSITORIES: biostudies-literature

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Structural basis for the aldolase and epimerase activities of Staphylococcus aureus dihydroneopterin aldolase.

Blaszczyk Jaroslaw J Li Yue Y Gan Jianhua J Yan Honggao H Ji Xinhua X

Journal of molecular biology 20070209 1

Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) and the epimerization of DHNP to 7,8-dihydromonopterin (DHMP). Although crystal structures of the enzyme from several microorganisms have been reported, no structural information is available about the critical interactions between DHNA and the trihydroxypropyl moiety of the substrate, which undergoes bond cleavage and formation. Here, we present the structures of St ...[more]

PMID: 17331536

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Project description:BackgroundDihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin and also the epimerization of DHNP to 7,8-dihydromonapterin. Previously, we determined the crystal structure of Staphylococcus aureus DHNA (SaDHNA) in complex with the substrate analogue neopterin (NP). We also showed that Escherichia coli DHNA (EcDHNA) and SaDHNA have significantly different binding and catalytic properties by biochemical analysis. On the basis of these structural and functional data, we proposed a catalytic mechanism involving two proton wires.ResultsTo understand the structural basis for the biochemical differences and further investigate the catalytic mechanism of DHNA, we have determined the structure of EcDHNA complexed with NP at 1.07-Å resolution [PDB:2O90], built an atomic model of EcDHNA complexed with the substrate DHNP, and performed molecular dynamics (MD) simulation analysis of the substrate complex. EcDHNA has the same fold as SaDHNA and also forms an octamer that consists of two tetramers, but the packing of one tetramer with the other is significantly different between the two enzymes. Furthermore, the structures reveal significant differences in the vicinity of the active site, particularly in the loop that connects strands β3 and β4, mainly due to the substitution of nearby residues. The building of an atomic model of the complex of EcDHNA and the substrate DHNP and the MD simulation of the complex show that some of the hydrogen bonds between the substrate and the enzyme are persistent, whereas others are transient. The substrate binding model and MD simulation provide the molecular basis for the biochemical behaviors of the enzyme, including noncooperative substrate binding, indiscrimination of a pair of epimers as the substrates, proton wire switching during catalysis, and formation of epimerization product.ConclusionsThe EcDHNA and SaDHNA structures, each in complex with NP, reveal the basis for the biochemical differences between EcDHNA and SaDHNA. The atomic substrate binding model and MD simulation offer insights into substrate binding and catalysis by DHNA. The EcDHNA structure also affords an opportunity to develop antimicrobials specific for Gram-negative bacteria, as DHNAs from Gram-negative bacteria are highly homologous and E. coli is a representative of this class of bacteria.

Project description:The biosynthesis of membrane lipids is an essential pathway for virtually all bacteria. Despite its potential importance for the development of novel antibiotics, little is known about the underlying signaling mechanisms that allow bacteria to control their membrane lipid composition within narrow limits. Recent studies disclosed an elaborate feed-forward system that senses the levels of malonyl-CoA and modulates the transcription of genes that mediate fatty acid and phospholipid synthesis in many Gram-positive bacteria including several human pathogens. A key component of this network is FapR, a transcriptional regulator that binds malonyl-CoA, but whose mode of action remains enigmatic. We report here the crystal structures of FapR from Staphylococcus aureus (SaFapR) in three relevant states of its regulation cycle. The repressor-DNA complex reveals that the operator binds two SaFapR homodimers with different affinities, involving sequence-specific contacts from the helix-turn-helix motifs to the major and minor grooves of DNA. In contrast with the elongated conformation observed for the DNA-bound FapR homodimer, binding of malonyl-CoA stabilizes a different, more compact, quaternary arrangement of the repressor, in which the two DNA-binding domains are attached to either side of the central thioesterase-like domain, resulting in a non-productive overall conformation that precludes DNA binding. The structural transition between the DNA-bound and malonyl-CoA-bound states of SaFapR involves substantial changes and large (>30 Å) inter-domain movements; however, both conformational states can be populated by the ligand-free repressor species, as confirmed by the structure of SaFapR in two distinct crystal forms. Disruption of the ability of SaFapR to monitor malonyl-CoA compromises cell growth, revealing the essentiality of membrane lipid homeostasis for S. aureus survival and uncovering novel opportunities for the development of antibiotics against this major human pathogen.

Dataset Information

Structural basis for the aldolase and epimerase activities of Staphylococcus aureus dihydroneopterin aldolase.

Publications

Structural basis for the aldolase and epimerase activities of Staphylococcus aureus dihydroneopterin aldolase.

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