John Pierce

Crystal structure of scytalone dehydratase--a disease determinant of the rice pathogen, Magnaporthe grisea.

BACKGROUND Rice blast is caused by the pathogenic fungus,-Magnaporthe grisea. Non-pathogenic mutants have been identified that lack enzymes in the biosynthetic pathway of dihydroxynapthalene-derived melanin. These enzymes are therefore prime targets for fungicides designed to control rice blast disease. One of the enzymes identified by genetic analysis as a disease determinant is scytalone dehydratase. RESULTS The three-dimensional structure of scytalone dehydratase in complex with a competitive inhibitor has been determined at 2.9 A resolution. A novel fold, a cone-shaped alpha + beta barrel, is adopted by the monomer in this trimeric protein, burying the hydrophobic active site in its interior. The interactions of the inhibitor with the protein side chains have been identified. The similarity of the inhibitor to the substrate and the side chains involved in binding afford some insights into possible catalytic mechanisms. CONCLUSIONS These results provide a first look into the structure and catalytic residues of a non-metal dehydratase, a large class of hitherto structurally uncharacterized enzymes. It is envisaged that a detailed structural description of scytalone dehydratase will assist in the design of new inhibitors for controlling rice blast disease.

The sites for catalysis and activation of ribulosebisphosphate carboxylase share a common domain.

The complexation of ribulosebiphosphate carboxylase with CO2, Mg2+, and carboxyarabinitol bisphosphate (CABP) to produce the quaternary enzyme-carbamate-Mg2+-CABP complex closely mimics the formation of the catalytically competent enzyme-carbamate-Mg2+-3-keto-CABP form during enzymatic catalysis. Quaternary complexes were prepared with various metals (Mg2+, Cd2+, Mn2+, Co2+, and Ni2+) and with specifically 13C-enriched ligands. 31P and 13C NMR studies of these complexes demonstrate that the activator CO2 site (carbamate site), the metal binding site, and the substrate binding site are contiguous. It follows that both the carboxylase and oxygenase activities of this bifunctional enzyme are influenced by the structures of the catalytic and activation sites.

Photosynthesis and photorespiration in a mutant of the cyanobacterium synechocystis pcc 6803 lacking carboxysomes

A mutant of the cyanobacterium Synechocystis PCC 6803 was obtained by replacing the gene of the carboxylation enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) with that of the photosynthetic bacterium Rhodospirillum rubrum. This mutant consequently lacks carboxysomes — the protein complexes in which the original enzyme is packed. It is incapable of growing at atmospheric CO2 levels and has an apparent photosynthetic affinity for inorganic carbon (Ci) which is 1000 times lower than that of the wild type, yet it accumulates more Ci than the wild type. The mutant appears to be defective in its ability to utilize the intracellular Ci pool for photosynthesis. Unlike the carboxysomal carboxylase activity of Rubisco, which is almost insensitive to inhibition by O2 in vitro, the soluble enzyme is competitively inhibited by O2. The photosynthetic rate and Ci compensation point of the wild type were hardly affected by low O2 levels. Above 100 μM O2, however, both parameters became inhibited. The CO2 compensation point of the mutant was linearly dependent on O2 concentration. The higher sensitivity of the mutant to O2 inhibition than that expected from in-vitro kinetics parameters of Rubisco, indicates a low capacity to recycle photorespiratory metabolites to Calvin-cycle intermediates.

Uptake and utilization of inorganic carbon by cyanobacteria

In the cyanobacteria, mechanisms exist that allow photosynthetic CO2 reduction to proceed efficiently even at very low levels of inorganic carbon. These inducible, active transport mechanisms enable the cyanobacteria to accumulate large internal concentrations of inorganic carbon that may be up to 1000-fold higher than the external concentration. As a result, the external concentration of inorganic carbon required to saturate cyanobacterial photosynthesis in vivo is orders of magnitude lower than that required to saturate the principal enzyme (ribulose bisphosphate carboxylase) involved in the fixation reactions. Since CO2 is the substrate for carbon fixation, the cyanobacteria somehow perform the neat trick of concentrating this small, membrane permeable molecule at the site of CO2 fixation. In this review, we will describe the biochemical and physiological experiments that have outlined the phenomenon of inorganic carbon accumulation, relate more recent genetic and molecular biological observations that attempt to define the constituents involved in this process, and discuss a speculative theory that suggests a unified view of inorganic carbon utilization by the cyanobacteria.

A diazine heterocycle replaces a six-membered hydrogen-bonded array in the active site of scytalone dehydratase

Abstract 4-aminoquinazoline II is a fully functional substitute for salicylamide I as a competitive inhibitor of scytalone dehydratase. Kinetic measurements demonstrate that both inhibitors bind to the enzyme in their neutral form, suggesting that the hydrogen-bonded array in I binds to the enzyme active site as an intact structural element.

NUCLEOTIDE SEQUENCE OF THE GENES ENCODING THE TWO MAJOR PROTEINS IN THE CYTOPLASMIC MEMBRANE OF SYNECHOCOCCUS PCC 7942

Cells of Synechococcus PCC 7942 contain 45-kD and 42-kD proteins as major proteins in the cytoplasmic membrane when grown under low CO2 conditions with nitrate as the nitrogen source [1]. Ammonium-grown cells lack the 45-kD protein [2,3,4], while high CO2-grown cells lack the 42-kD protein [5]. Insertional mutation of the gene for the 45-kD protein resulted in inactivation of nitrate transport, indicating that the protein is an essential component of the nitrate-transporting mechanism [4]. The 42-kD protein, on the other hand, was once supposed to be involved in the transport of inorganic carbon (CO2 or HCO 3 − ) [5]. However, inactivation of the gene for this protein did not affect the activities of CO2/HCO 3 − transport in Synechococcus [6], and the role of the 42-kD protein remains unclear.

Some Mechanistic Aspects of Ribulose Bisphosphate Carboxylase

Water is involved in the carboxylase reaction in 2 distinct ways (Fig 1.) Besides its stoichiometric involvement, it hydrates both substrates. While carbon dioxide has long been recognised as the active species, no information is yet available on how the enzyme handles the geM-diol form of RuBP. The equilibria and kinetics of interconversion of the keto and gem-diol forms of RuBP have been studied by NMR and UV spectroscopy. As Table I indicates the equilibrium is temperature dependent, higher temperatures favoring the keto form.