Cofactor bypass variants reveal a conformational control mechanism governing cell wall polymerase activity.

To fortify their cytoplasmic membrane and protect it from osmotic rupture, most bacteria surround themselves with a peptidoglycan (PG) exoskeleton synthesized by the penicillin-binding proteins (PBPs). As their name implies, these proteins are the targets of penicillin and related antibiotics. We and others have shown that the PG synthases PBP1b and PBP1a of Escherichia coli require the outer membrane lipoproteins LpoA and LpoB, respectively, for their in vivo function. Although it has been demonstrated that LpoB activates the PG polymerization activity of PBP1b in vitro, the mechanism of activation and its physiological relevance have remained unclear. We therefore selected for variants of PBP1b (PBP1b*) that bypass the LpoB requirement for in vivo function, reasoning that they would shed light on LpoB function and its activation mechanism. Several of these PBP1b variants were isolated and displayed elevated polymerization activity in vitro, indicating that the activation of glycan polymer growth is indeed one of the relevant functions of LpoB in vivo. Moreover, the location of amino acid substitutions causing the bypass phenotype on the PBP1b structure support a model in which polymerization activation proceeds via the induction of a conformational change in PBP1b initiated by LpoB binding to its UB2H domain, followed by its transmission to the glycosyl transferase active site. Finally, phenotypic analysis of strains carrying a PBP1b* variant revealed that the PBP1b-LpoB complex is most likely not providing an important physical link between the inner and outer membranes at the division site, as has been previously proposed.

A comparative structural analysis reveals distinctive features of co-factor binding and substrate specificity in plant aldo-keto reductases.

Plant aldo-keto reductases of the AKR4C subfamily play key roles during stress and are attractive targets for developing stress-tolerant crops. However, these AKR4Cs show little to no activity with previously-envisioned sugar substrates. We hypothesized a structural basis for the distinctive cofactor binding and substrate specificity of these plant enzymes. To test this, we solved the crystal structure of a novel AKR4C subfamily member, the AKR4C7 from maize, in the apo form and in complex with NADP(+). The binary complex revealed an intermediate state of cofactor binding that preceded closure of Loop B, and also indicated that conformational changes upon substrate binding are required to induce a catalytically-favorable conformation of the active-site pocket. Comparative structural analyses of homologues (AKR1B1, AKR4C8 and AKR4C9) showed that evolutionary redesign of plant AKR4Cs weakened interactions that stabilize the closed conformation of Loop B. This in turn decreased cofactor affinity and altered configuration of the substrate-binding site. We propose that these structural modifications contribute to impairment of sugar reductase activity in favor of other substrates in the plant AKR4C subgroup, and that catalysis involves a three-step process relevant to other AKRs.

Mechanistic implications from structures of yeast alcohol dehydrogenase complexed with coenzyme and an alcohol.

Yeast alcohol dehydrogenase I is a homotetramer of subunits with 347 amino acid residues, catalyzing the oxidation of alcohols using NAD(+) as coenzyme. A new X-ray structure was determined at 3.0 Å where both subunits of an asymmetric dimer bind coenzyme and trifluoroethanol. The tetramer is a pair of back-to-back dimers. Subunit A has a closed conformation and can represent a Michaelis complex with an appropriate geometry for hydride transfer between coenzyme and alcohol, with the oxygen of 2,2,2-trifluoroethanol ligated at 2.1 Å to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. Subunit B has an open conformation, and the coenzyme interacts with amino acid residues from the coenzyme binding domain, but not with residues from the catalytic domain. Coenzyme appears to bind to and dissociate from the open conformation. The catalytic zinc in subunit B has an alternative, inverted coordination with Cys-43, Cys-153, His-66 and the carboxylate of Glu-67, while the oxygen of trifluoroethanol is 3.5 Å from the zinc. Subunit B may represent an intermediate in the mechanism after coenzyme and alcohol bind and before the conformation changes to the closed form and the alcohol oxygen binds to the zinc and displaces Glu-67.

Human FAD synthase is a bi-functional enzyme with a FAD hydrolase activity in the molybdopterin binding domain.

FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is involved in the biochemical pathway for converting riboflavin into FAD. Human FADS exists in different isoforms. Two of these have been characterized and are localized in different subcellular compartments. hFADS2 containing 490 amino acids shows a two domain organization: the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and a resembling molybdopterin-binding (MPTb) domain. By a multialignment of hFADS2 with other MPTb containing proteins of various organisms from bacteria to plants, the critical residues for hydrolytic function were identified. A homology model of the MPTb domain of hFADS2 was built, using as template the solved structure of a T. acidophilum enzyme. The capacity of hFADS2 to catalyse FAD hydrolysis was revealed. The recombinant hFADS2 was able to hydrolyse added FAD in a Co(2+) and mersalyl dependent reaction. The recombinant PAPS reductase domain is not able to perform the same function. The mutant C440A catalyses the same hydrolytic function of WT with no essential requirement for mersalyl, thus indicating the involvement of C440 in the control of hydrolysis switch. The enzyme C440A is also able to catalyse hydrolysis of FAD bound to the PAPS reductase domain, which is quantitatively converted into FMN.

Crystal structure of steroid reductase SRD5A reveals conserved steroid reduction mechanism.

Steroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ4 structure, such as testosterone or progesterone, are catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. SRD5A2 is already a target of clinically relevant drugs. However, the detailed mechanism of SRD5A-mediated reduction remains elusive. Here we report the crystal structure of PbSRD5A from Proteobacteria bacterium, a homolog of both SRD5A1 and SRD5A2, in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic substrate binding cavity, whereas TM5-7 coordinate cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with biochemical characterization, define the substrate binding pocket of SRD5As, explain the properties of disease-related mutants and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible.

Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors.

Recent history is punctuated by the emergence of highly pathogenic coronaviruses such as SARS- and MERS-CoV into human circulation. Upon infecting host cells, coronaviruses assemble a multi-subunit RNA-synthesis complex of viral non-structural proteins (nsp) responsible for the replication and transcription of the viral genome. Here, we present the 3.1 Å resolution structure of the SARS-CoV nsp12 polymerase bound to its essential co-factors, nsp7 and nsp8, using single particle cryo-electron microscopy. nsp12 possesses an architecture common to all viral polymerases as well as a large N-terminal extension containing a kinase-like fold and is bound by two nsp8 co-factors. This structure illuminates the assembly of the coronavirus core RNA-synthesis machinery, provides key insights into nsp12 polymerase catalysis and fidelity and acts as a template for the design of novel antiviral therapeutics.

Methanogenic heterodisulfide reductase (HdrABC-MvhAGD) uses two noncubane [4Fe-4S] clusters for reduction.

In methanogenic archaea, the carbon dioxide (CO2) fixation and methane-forming steps are linked through the heterodisulfide reductase (HdrABC)-[NiFe]-hydrogenase (MvhAGD) complex that uses flavin-based electron bifurcation to reduce ferredoxin and the heterodisulfide of coenzymes M and B. Here, we present the structure of the native heterododecameric HdrABC-MvhAGD complex at 2.15-angstrom resolution. HdrB contains two noncubane [4Fe-4S] clusters composed of fused [3Fe-4S]-[2Fe-2S] units sharing 1 iron (Fe) and 1 sulfur (S), which were coordinated at the CCG motifs. Soaking experiments showed that the heterodisulfide is clamped between the two noncubane [4Fe-4S] clusters and homolytically cleaved, forming coenzyme M and B bound to each iron. Coenzymes are consecutively released upon one-by-one electron transfer. The HdrABC-MvhAGD atomic model serves as a structural template for numerous HdrABC homologs involved in diverse microbial metabolic pathways.

Preparation, characterization and evaluation of coenzyme Q10 binary solid dispersions for enhanced solubility and dissolution.

Phase solubility behavior of coenzyme Q10 (CoQ10) at 25 degrees C in various molar solutions of poloxamer 188 (P188) in water was observed and their binary solid dispersions (BSD) at different weight ratios were prepared by a simple, rapid, cost effective, uncomplicated and potentially scalable low temperature melting method. BSDs were characterized by scanning electron microscopy (SEM) and differential scanning calorimetry (DSC), and evaluated for improved solubility at 25 degrees C and 37 degrees C and in-vitro release of CoQ10 at 37 degrees C in distilled water. Solubility of CoQ10 increased with increasing concentrations of P188 in water. Gibbs free energy (deltaG(o)tr) values were all negative indicating the spontaneous nature of CoQ10 solubilization and decreased with increasing concentration of P188 demonstrating that the reaction conditions became more favorable as the concentration of P188 increased. DSC and SEM analysis indicated that the homogeneity of dispersion was not at the molecular level. However, BSDs exhibited a remarkably improved aqueous solubility and dissolution of CoQ10.

Colocalization of muscle FBPase and muscle aldolase on both sides of the Z-line.

Previously we have reported that in vitro muscle aldolase binds to muscle FBPase [Biochem. Biophys. Res. Commun. 275 (2000) 611-616] which results in the changes of regulatory properties of the latter enzyme. In the present paper, the evidence that aldolase binds to FBPase in living cell is presented. The colocalization experiment, in which aldolase was diffused into skinned fibres that had been pre-incubated with FBPase, has shown that aldolase in the presence of FBPase binds predominantly to the Z-line. The existence of a triple aldolase-FBPase-alpha-actinin complex was confirmed through a real-time interaction analysis using the BIAcore biosensor. The colocalization of FBPase and aldolase on alpha-actinin of the Z-line indicates the existence of glyconeogenic metabolon in vertebrates' myocytes.