Peter W. Goodenough

Germin is a manganese containing homohexamer with oxalate oxidase and superoxide dismutase activities

Germin is a hydrogen peroxide generating oxalate oxidase with extreme thermal stability; it is involved in the defense against biotic and abiotic stress in plants. The structure, determined at 1.6 Å resolution, comprises β-jellyroll monomers locked into a homohexamer (a trimer of dimers), with extensive surface burial accounting for its remarkable stability. The germin dimer is structurally equivalent to the monomer of the 7S seed storage proteins (vicilins), indicating evolution from a common ancestral protein. A single manganese ion is bound per germin monomer by ligands similar to those of manganese superoxide dismutase (MnSOD). Germin is also shown to have SOD activity and we propose that the defense against extracellular superoxide radicals is an important additional role for germin and related proteins.

Rational Conversion of Substrate and Product Specificity in a Salvia Monoterpene Synthase: Structural Insights into the Evolution of Terpene Synthase Function

Terpene synthases are responsible for the biosynthesis of the complex chemical defense arsenal of plants and microorganisms. How do these enzymes, which all appear to share a common terpene synthase fold, specify the many different products made almost entirely from one of only three substrates? Elucidation of the structure of 1,8-cineole synthase from Salvia fruticosa (Sf-CinS1) combined with analysis of functional and phylogenetic relationships of enzymes within Salvia species identified active-site residues responsible for product specificity. Thus, Sf-CinS1 was successfully converted to a sabinene synthase with a minimum number of rationally predicted substitutions, while identification of the Asn side chain essential for water activation introduced 1,8-cineole and α-terpineol activity to Salvia pomifera sabinene synthase. A major contribution to product specificity in Sf-CinS1 appears to come from a local deformation within one of the helices forming the active site. This deformation is observed in all other mono- or sesquiterpene structures available, pointing to a conserved mechanism. Moreover, a single amino acid substitution enlarged the active-site cavity enough to accommodate the larger farnesyl pyrophosphate substrate and led to the efficient synthesis of sesquiterpenes, while alternate single substitutions of this critical amino acid yielded five additional terpene synthases.

Plastid changes during the conversion of chloroplasts to chromoplasts in ripening tomatoes

Methods were developed for the isolation of plastids from mature green and ripening tomatoes (Lycopersicon esculentum Mill.) and purification by sucrose or Percoll density-gradient centrifugation. Assessment of the purity of preparations involved phase-contrast and electron microscopy, assays for marker enzymes and RNA extraction and analysis. Proteins were extracted from isolated plastids at different ripening stages and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The profiles obtained from chloroplasts and chromoplasts showed many qualitative and quantitative differences. Labelling of proteins with [35S]methionine in vivo showed that there was active protein synthesis throughout ripening, but there was a change in the plastid proteins made as ripening proceeded. The cellular location of synthesis of specific proteins has yet to be established.

A study of the heat stabilities of a number of indigenous milk enzymes

Heat stability profiles of a number of indigenous bovine milk enzymes were examined with the object of being able to monitor heat treatments slightly more severe than typical pasteurization conditions by measurements of residual enzyme activity after heating. Assay procedures were limited to simple fluorimetric, or preferably colorimetric, methods that would be most likely to form the basis of a quick, simple and inexpensive test. Both lipoprotein lipase and α-fucosidase were relatively sensitive to heat and were totally inactivated at temperature/time combinations below those of pasteurization, but the latter may be satisfactory for studying temperatures in the range 55–65°C. Rather more heat stable were N -acetyl-β-glucosaminidase and γ-glutamyl transpeptidase, which may be most appropriate for 65–75°C and 70–80°C respectively. Higher temperature treatments between 80 and 90°C could best be investigated by following α-mannosidase or xanthine oxidase activity.

Barley oxalate oxidase is a hexameric protein related to seed storage proteins: evidence from X‐ray crystallography

The oxalate oxidase enzyme expressed in barley roots is a thermostable, protease‐resistant enzyme that generates H2O2. It has great medical importance because of its use to assay plasma and urinary oxalate, and it has also been used to generate transgenic, pathogen‐resistant crops. This protein has now been purified and three types of crystals grown. X‐ray analysis shows that the symmetry present in these crystals is consistent with a hexameric arrangement of subunits, probably a trimer of dimers. This structure may be similar to that found in the related seed storage proteins.

NADP-linked malic enzyme and malate metabolism in ageing tomato fruit

Abstract Malate dehydrogenase (oxaloacetate-decarboxylating) (NADP + ) EC 1.1.1.40, malic enzyme, has been purified 40-fold to a homogeneous state using affinity chromatography and gel permeation chromatography. The M r is 260–265 K with four subunits each of 64–65 K. The enzyme has some competitive or non-competitive inhibitors, particularly some of the Krebs cycle acids and exhibits a rapid rise in activity at the same time as activity of the enzymes of the Krebs cycle are decreasing in the tomato mitochrondrion. The malic enzyme is restricted to the cytosol. The relevance of this information to malate metabolism in plants is discussed.

Changes in colour, polygalacturonase monosaccharides and organic acids during storage of tomatoes

Abstract The appearance of polygalacturonase and red pigmentation in mature-green tomato fruit was prevented by storing the fruit in 5% O 2 , 5% CO 2 and 90% N 2 . However, the breakdown of starch to give monosaccharides and the change in concentration of organic acids which is normally associated with ripening still took place. On removal of the fruit to ambient conditions, polygalacturonase was synthesized and the fruit changed colour but monosaccharide and organic acid concentrations did not change.

Nucleotide sequence and expression in Escherichia coli of cDNAs encoding papaya proteinase omega from Carica papaya.

We have cloned and sequenced two similar, but distinct, cDNAs from both fruit and leaf tissues of Carica papaya. The C-terminal portion of the predicted amino acid (aa) sequence of one of the clones has complete identity with the mature enzyme sequence of the cysteine proteinase papaya proteinase omega (Pp omega). The second clone contains ten individual bp changes compared with the first and encodes a protein with three single-aa substitutions, only one of which is located in the mature sequence, but most noticeably carries an additional 19-aa C-terminal extension. The clones encode pre-pro precursor isoforms of Pp omega. The former of these clones has been expressed in Escherichia coli using a T7 polymerase expression system to produce insoluble pro-enzyme which has been solubilized and refolded to yield auto-activable pro-Pp omega.

Crystal structure of a caricain D158E mutant in complex with E-64.

The structure of the D158E mutant of caricain (previously known as papaya protease omega) in complex with E‐64 has been determined at 2.0 Å resolution (overall R factor 19.3%). The structure reveals that the substituted glutamate makes the same pattern of hydrogen bonds as the aspartate in native caricain. This was not anticipated since in the native structure there is insufficient room to accommodate the glutamate side chain. The glutamate is accommodated in the mutant by a local expansion of the structure demonstrating that small structural changes are responsible for the change in activity.

Isolation of four major subunits from Clostridium thermocellum cellulosome and their synergism in the hydrolysis of crystalline cellulose.

The cellulosome of Clostridium thermocellum, purified by affinity chromatography, was dissociated under mild conditions and separated by SDS-PAGE. Two major p-nitrophenylcellobiosidases (PNPCases I and II) corresponding to the S5 (103 kDa) and S8 (78 kDa) subunits and one major carboxymethylcellulase (CMCase) coinciding with the S11 (60.5 kDa) subunit were isolated and characterized using carboxymethylcellulose (CMC), H3PO4-swollen cellulose and cello-oligosaccharides. Both PNPCases showed little effect on the viscosity of CMC and released twice as much total sugar as reducing sugar from H3PO4-swollen cellulose. The CMCase released ten times more total sugar than reducing sugar from H3PO4-swollen cellulose and reduced the viscosity of CMC rapidly. None of these enzymes was active on cellotriose. Both PNPCases released cellobiose from cellotetraose, and cellobiose and cellotriose from cellopentaose. In contrast, CMCase was active only on cellopentaose and released mainly glucose. Use of MeUmb(Glc)n revealed that both PNPCases cleaved preferentially either the second or fourth linkage from the non-reducing end while the CMCase was specific for the internal glycosidic bonds. Thus, the PNPCases and CMCase behaved as typical exo- and endoglucanases, respectively. When tested individually, all three enzymes degraded Avicel only to a small extent. A 1.5-2.0-fold increase in sugar release was observed when CMCase was combined with either PNPCase I, II or both. Combining S1 with either PNPCase II or CMCase resulted in fourfold synergism in the hydrolysis of Avicel. Synergism was sevenfold when all three enzymes were combined with S1.