Apparent unbalance between the activities of 6-phosphogluconate and glucose-6-phosphate dehydrogenases in rat liver

The ratio of activities of 6-phosphogluconate dehydrogenase/glucose-6-phosphate dehydrogenase measured in liver extracts of rats in lipogenic nutritional conditions is only 0.2, suggesting an apparent physiological unbalance between the two dehydrogenases of the hexosemonophosphate shunt. This potential unbalance is enhanced by the fact that TPNH is a more powerful competitive inhibitor of 6-phosphogluconate dehydrogenase than of glucose-6-phosphate dehydrogenase. Accordingly, a strong activation of 6-phosphogluconate dehydrogenase would be required for efficient functioning of this pathway, unless there is an alternative outlet for 6-phosphogluconate so far unrecognized in animal tissues.     

Modular organization of FDH: Exploring the basis of hydrolase catalysis

An abundant enzyme of liver cytosol, 10-formyltetrahydrofolate dehydrogenase (FDH), is an interesting example of a multidomain protein. It consists of two functionally unrelated domains, an aldehyde dehydrogenase-homologous domain and a folate-binding hydrolase domain, which are connected by an approximately 100-residue linker. The amino-terminal hydrolase domain of FDH (Nt-FDH) is a homolog of formyl transferase enzymes that utilize 10-formyl-THF as a formyl donor. Interestingly, the concerted action of all three domains of FDH produces a new catalytic activity, NADP+-dependent oxidation of 10-formyltetrahydrofolate (10-formyl-THF) to THF and CO2. The present studies had two objectives: First, to explore the modular organization of FDH through the production of hybrid enzymes by domain replacement with methionyl-tRNA formyltransferase (FMT), an enzyme homologous to the hydrolase domain of FDH. The second was to explore the molecular basis for the distinct catalytic mechanisms of Nt-FDH and related 10-formyl-THF utilizing enzymes. Our studies revealed that FMT cannot substitute for the hydrolase domain of FDH in order to catalyze the dehydrogenase reaction. It is apparently due to inability of FMT to catalyze the hydrolysis of 10-formyl-THF in the absence of the cosubstrate of the transferase reaction despite the high similarity of the catalytic centers of the two enzymes. Our results further imply that Ile in place of Asn in the FDH hydrolase catalytic center is an important determinant for hydrolase catalysis as opposed to transferase catalysis.     

<Emphasis Type="Italic"> Cis</Emphasis>-acting elements sufficient for induction of<Emphasis Type="Italic"> FDH1</Emphasis> expression by formate in the methylotrophic yeast<Emphasis Type="Italic"> Candida boidinii</Emphasis>

The FDH1 gene of Candida boidinii encodes an NAD⁺-dependent formate dehydrogenase, which catalyzes the last reaction in the methanol dissimilation pathway. FDH1 expression is strongly induced by methanol, as are the promoters of the genes AOD1 (alcohol oxidase) and DAS1 (dihydroxyacetone synthase). FDH1 expression can be induced by formate when cells are grown on a medium containing glucose as a carbon source, whereas expression of AOD1 and DAS1 is completely repressed in the presence of glucose. Using deletion analyses, we identified two cis-acting regulatory elements, termed UAS-FM and UAS-M, respectively, in the 5 non-coding region of the FDH1 gene. Both elements were necessary for full induction of the FDH1 promoter by methanol, while only the UAS-FM element was required for full induction by formate. Irrespective of whether induction was achieved with methanol or formate, the UAS-FM element enhanced the level of induction of the FDH1 promoter in a manner dependent on the number of copies, but independent of their orientation, and also converted the ACT1 promoter from a constitutive into an inducible element. Our results not only provide a powerful promoter for heterologous gene expression, but also yield insights into the mechanism of regulation of FDH1 expression at the molecular level.Communicated by C. P. Hollenberg     

Structural basis of substrate specificity in human glyoxylate reductase/hydroxypyruvate reductase

Human glyoxylate reductase/hydroxypyruvate reductase (GRHPR) is a D-2-hydroxy-acid dehydrogenase that plays a critical role in the removal of the metabolic by-product glyoxylate from within the liver. Deficiency of this enzyme is the underlying cause of primary hyperoxaluria type 2 (PH2) and leads to increased urinary oxalate levels, formation of kidney stones and renal failure. Here we describe the crystal structure of human GRHPR at 2.2 A resolution. There are four copies of GRHPR in the crystallographic asymmetric unit: in each homodimer, one subunit forms a ternary (enzyme+NADPH+reduced substrate) complex, and the other a binary (enzyme+NADPH) form. The spatial arrangement of the two enzyme domains is the same in binary and ternary forms. This first crystal structure of a true ternary complex of an enzyme from this family demonstrates the relationship of substrate and catalytic residues within the active site, confirming earlier proposals of the mode of substrate binding, stereospecificity and likely catalytic mechanism for these enzymes. GRHPR has an unusual substrate specificity, preferring glyoxylate and hydroxypyruvate, but not pyruvate. A tryptophan residue (Trp141) from the neighbouring subunit of the dimer is projected into the active site region and appears to contribute to the selectivity for hydroxypyruvate. This first crystal structure of a human GRHPR enzyme also explains the deleterious effects of naturally occurring missense mutations of this enzyme that lead to PH2.     

The comparative interaction of human and bovine serum albumins with CYP2C9 in human liver microsomes

The effect of human serum albumin (HSA), in its endogenous, free fatty acid free (FAF) and globulin free (GF) form, on the activity of CYP2C9 was studied in human liver microsomes using tolbutamide as the substrate. The widely used BSA was included to assess the differential effect of BSA and HSA. CYP2C9 activity was expressed as CLint (Vmax/Km). HSA(FAF) and BSA showed a concentration-dependent and biphasic (activation and inhibition) interaction with CYP2C9 activity. HSA(GF) and HSA exhibited an inhibitory effect, with an inhibition constant, Ki, of 19.9 microM (0.13% albumin) and 42.2 microM (0.35% albumin), respectively. Enzyme-kinetics revealed that the activation is accompanied by a decrease in Km values, while with inhibition Km values increased. A simplified method to calculate clearance, utilizing a single slope (V/S) determination based on V over the lowest linear range of [S] (designated as CLone) was assessed. Virtually identical values were obtained for CLint and CLone. The free-drug hypothesis was tested by comparing ratios of relative CLint/unbound fraction (FDH Test ratio). The FDH Test ratio for HSA was about 1, indicating that HSA binding of tolbutamide reduced the CYP2C9 activity in accord with the free-drug hypothesis. The FDH Test ratios for BSA and HSA(FAF) were 3.7 and 3.0, revealing a monophasic activation of CYP2C9. For 2%HSA(GF) the ratio of 0.3 confirmed inhibition. As revealed by their removal, free fatty acids and globulins, significantly alter the interaction of HSA with CYP2C9. In addition, HSA and BSA showed different effects on the oxidation of tolbutamide by CYP2C9.     

Chloramphenicol Biosynthesis: The Structure of CmlS, a Flavin-Dependent Halogenase Showing a Covalent Flavin-Aspartate Bond

Chloramphenicol is a halogenated natural product bearing an unusual dichloroacetyl moiety that is critical for its antibiotic activity. The operon for chloramphenicol biosynthesis in Streptomyces venezuelae encodes the chloramphenicol halogenase CmlS, which belongs to the large and diverse family of flavin-dependent halogenases (FDH’s). CmlS was previously shown to be essential for the formation of the dichloroacetyl group. Here we report the X-ray crystal structure of CmlS determined at 2.2 Å resolution, revealing a flavin monooxygenase domain shared by all FDHs, but also a unique ‘winged-helix’ C-terminal domain that creates a T-shaped tunnel leading to the halogenation active site. Intriguingly, the C-terminal tail of this domain blocks access to the halogenation active site, suggesting a structurally dynamic role during catalysis. The halogenation active site is notably nonpolar and shares nearly identical residues with Chondromyces crocatus tyrosyl halogenase (CndH), including the conserved Lys (K71) that forms the reactive chloramine intermediate. The exception is Y350, which could be used to stabilize enolate formation during substrate halogenation. The strictly conserved residue E44, located near the isoalloxazine ring of the bound flavin adenine dinucleotide (FAD) cofactor, is optimally positioned to function as a remote general acid, through a water-mediated proton relay, which could accelerate the reaction of the chloramine intermediate during substrate halogenation, or the oxidation of chloride by the FAD(C4α)-OOH intermediate. Strikingly, the 8α carbon of the FAD cofactor is observed to be covalently attached to D277 of CmlS, a residue that is highly conserved in the FDH family. In addition to representing a new type of flavin modification, this has intriguing implications for the mechanism of FDHs. Based on the crystal structure and in analogy to known halogenases, we propose a reaction mechanism for CmlS.     

Structural Evidence for the Functional Importance of the Heme Domain Mobility in Flavocytochrome b2

Yeast flavocytochrome b₂ (Fcb2) is an l-lactate:cytochrome c oxidoreductase in the mitochondrial intermembrane space participating in cellular respiration. Each enzyme subunit consists of a cytochrome b₅-like heme domain and a flavodehydrogenase (FDH) domain. In the Fcb2 crystal structure, the heme domain is mobile relative to the tetrameric FDH core in one out of two subunits. The monoclonal antibody B2B4, elicited against the holoenzyme, recognizes only the native heme domain in the holoenzyme. When bound, it suppresses the intramolecular electron transfer from flavin to heme b₂, hence cytochrome c reduction. We report here the crystal structure of the heme domain in complex with the Fab at 2.7 Å resolution. The Fab epitope on the heme domain includes the two exposed propionate groups of the heme, which are hidden in the interface between the domains in the complete subunit. The structure discloses an unexpected plasticity of Fcb2 in the neighborhood of the heme cavity, in which the heme has rotated. The epitope overlaps with the docking area of the FDH domain onto the heme domain, indicating that the antibody displaces the heme domain in a movement of large amplitude. We suggest that the binding sites on the heme domain of cytochrome c and of the FDH domain also overlap and therefore that cytochrome c binding also requires the heme domain to move away from the FDH domain, so as to allow electron transfer between the two hemes. Based on this hypothesis, we propose a possible model of the Fcb2·cytochrome c complex. Interestingly, this model shares similarity with that of the cytochrome b₅·cytochrome c complex, in which cytochrome c binds to the surface around the exposed heme edge of cytochrome b₅. The present results therefore support the idea that the heme domain mobility is an inherent component of the Fcb2 functioning.     

Mapping photoautotrophic metabolism with isotopically nonstationary (13)C flux analysis

Understanding in vivo regulation of photoautotrophic metabolism is important for identifying strategies to improve photosynthetic efficiency or re-route carbon fluxes to desirable end products. We have developed an approach to reconstruct comprehensive flux maps of photoautotrophic metabolism by computational analysis of dynamic isotope labeling measurements and have applied it to determine metabolic pathway fluxes in the cyanobacterium Synechocystis sp. PCC6803. Comparison to a theoretically predicted flux map revealed inefficiencies in photosynthesis due to oxidative pentose phosphate pathway and malic enzyme activity, despite negligible photorespiration. This approach has potential to fill important gaps in our understanding of how carbon and energy flows are systemically regulated in cyanobacteria, plants, and algae.