Structural and biochemical studies of GH family 12 cellulases: improved thermal stability,and ligand complexes

In this review we will describe how we have gathered structural and biochemical information from several homologous cellulases from one class of glycoside hydrolases (GH family 12), and used this information within the framework of a protein-engineering program for the design of new variants of these enzymes. These variants have been characterized to identify some of the positions and the types of mutations in the enzymes that are responsible for some of the biochemical differences in thermal stability and activity between the homologous enzymes. In this process we have solved the three-dimensional structure of four of these homologous GH 12 cellulases: Three fungal enzymes, Humicola grisea Cel12A, Hypocrea jecorina Cel12A and Hypocrea schweinitzii Cel12A, and one bacterial, Streptomyces sp. 11AG8 Cel12A. We have also determined the three-dimensional structures of the two most stable H. jecorina Cel12A variants. In addition, four ligand-complex structures of the wild-type H. grisea Cel12A enzyme have been solved and have made it possible to characterize some of the interactions between substrate and enzyme. The structural and biochemical studies of these related GH 12 enzymes, and their variants, have provided insight on how specific residues contribute to protein thermal stability and enzyme activity. This knowledge can serve as a structural toolbox for the design of Cel12A enzymes with specific properties and features suited to existing or new applications.     

Wampetin,a furocoumarin from Clausena wampi

A new furocoumarin wampetin has been isolated from Clausena wampi (syn. Clausena lansium). The structure was established from ¹H NMR, ¹³C NMR, MS and chemical data.     

A 34-seco-ambrosanolide from Ambrosia artemisiifolia

A new 3,4-seco-ambrosanolide was isolated from Ambrosia artemisiifolia and identified by means of spectroscopic evidence ¹H and ¹³C NMR, IR and MS).     

Biosynthesis of steroidal withanolides in Withania somnifera

Administration of 24-methylene-cholesterol-[28-³H] to Withania somnifera, yielded [³H] radioactivity in the isolated withaferin A and withanolide D, whereas administered 24-(R,S)-methyl-cholesterol-[28-³H] was not incorporated into these compounds. 24-Methylene-cholesterol is, therefore, proposed as a sterol precursor of the withanolides. A novel procedure is described for the isolation of withanolides from W. somnifera. This method in conjunction with an improved procedure for administration of labelled sterols and mevalonolactone produces a greatly increased yield of labelled withanolides.     

Insect antifeedant secoaromadendrane-type sesquiterpenes from Plagiochila species

The characteristic pungency of the liverworts Plagiochila species P. fruticosa, P. hattoriana, P. ovalifolia and P. yokogurensis is due to a new ent-secoaromadendrane-type sesquiterpene hemiacetal, plagiochiline A, which exhibits very strong antifeedant activity against the African army worm, Spodoptera exempta at 1–10ng/cm². Two new secoaromadendranes, plagiochilide and furanoplagiochilal A, together with the previously known plagiochiline C were isolated from P. yokogurensis. Plagiochilal A, which may be a precursor of plagiochilide and its related hemiacetals, and a bitter principle, plagiochiline B were also isolated from P. hattoriana. P. ovalifolia contained plagiochilines A, B and C. From P. fruticosa, plagiochilide and plagiochilines A, B and C were isolated. The structures of the new secoaromadendrane-type sesquiterpenes were elucidated by extensive ¹H NMR and ¹³CNMR studies.     

Catharinensine,an oxindole alkaloid from Peschiera catharinensis

The structure of catharinensine isolated from Peschiera catharinensis was deduced from a detailed analysis of its ¹H NMR, ¹³C NMR and mass spectra. The synthesis of 17-demethoxyrhynchophylline confirmed its structure, and led us to suggest the configuration allo A for catharinensine. Coronaridine, isovoacangine, heyneanine, 16-epiaffinine, decarbomethoxyvoacamine and conodurine were also isolated.     

Sarmentosin epoxide,a new cyanogenic compound from Sedum cepaea

A new cyanogenic compound, sarmentosin epoxide, was isolated from the aerial parts of Sedum cepaea. Its structure was established mainly by ¹H     

Four aromatic derivatives from Ruta angustifolia

A new group of four shikimate metabolites have been isolated from aerial parts of Ruta angustifolia and their structures deduced from IR, ¹H NMR, ¹³C NMR and MS data. They have been named moskachans.     

Teupolin III,a furanoid diterpene from Teucrium polium

A new clerodane derivative was isolated from Teucrium polium var. polium. The structure and stereochemistry have been established by detailed studies of the ¹H NMR spectra.     

11,13-Dehydrodesacetylmatricarin and other sesquiterpene lactones from Artemisia ludoviciana var. Ludoviciana and the identity of artecanin and chyrsartemin B

11,13-Dehydrodesacetylmatricarin, achillin, parishin-C, vulgarin and artecanin were isolated from A. ludoviciana var. ludoviciana. The identity of artecanin and chrysartemin-B was confirmed and ¹³ C NMR and ¹H NMR data are described.     

Three new hetisine-type diterpenoid alkaloids from Aconitum coreanum

Three new hetisine-type diterpenoid alkaloids, Guan-fu base V (GFV, 1), Guan-fu base W (GFW, 2) and Guan-fu base X (GFX, 3) were isolated from the roots of Aconitum coreanum (Lèvl.) Rapaics. Their structures were established by direct interpretation of their spectral data, mainly high resolution electrospray ionization mass spectrometry (HR-ESI-MS), ¹D and ²D NMR (¹H–¹H COSY, ROESY, HSQC and HMBC). GFX is the third N-oxygenated hetisine-type diterpenoid alkaloid isolated from A. coreanum.     

Acalyphin,a cyanogenic glucoside from Acalypha indica

A new cyanogenic glucoside, acalyphin, was isolated from the aerial parts of Acalypha indica and its structure identified mainly by ¹H NMR and     

6,8-Di-C-methyldihydromyricetin from Alluaudia humbertii

A novel flavanonol isolated from bark and spines of Alluaudia humbertii has been identified as 6,8-di-C-methyldihydromyricetin, by means of UV, mass and ¹H NMR spectra.     

E-2(S)-amino-3-methyl-3-pentenoic acid from coniogramme intermedia

A new amino acid, E-2(S)-amino-3-methyl-3-pentenoic acid was isolated from Coniogramme intermedia. The structure was elucidated by elementary analysis, optical rotation, catalytic hydrogenation, ¹H and ¹³C NMR spectra.     

Glycosylation of nascent polypeptide chains in Aspergillus niger

Polysomes were isolated from Aspergillus niger and were characterized on sucrose gradients in several ways. First, they were found to be susceptible to degradation by treatment with RNase or EDTA. Second, they were labeled after treating mycelia with short pulses of [³H]uridine or [³H]leucine prior to polysome isolation. Third, they were capable of stimulating incorporation of [³H]leucine into trichloroacetic acid-precipitable material in a chick reticulocyte cell-free protein-synthesizing system. When isolated [³H]leucine pulse-labeled polysomes were treated with either EDTA-RNase or puromycin, 80–90% of the radioactivity was released, indicating that only the nascent polypeptide chains were labeled. After exposing mycelia for 1 min to [¹⁴C]mannose, the polysomes were exclusively labeled, indicating that initial glycosylation takes place on nascent polypeptide chains. Preincubation of mycelia with 2-deoxyglucose followed by pulse-labeling with [³H]leucine and [¹⁴C]mannose showed that 2-deoxy-d-glucose inhibits both protein synthesis and glycosylation. However, similar preincubation with tunicamycin caused an 80% drop in [¹⁴C]mannose label in the polysomes, but only a 10–20% drop of [³H]leucine label, suggesting that glycosylation of nascent chains in A. niger involves an oligosaccharide-lipid intermediate, since it has been shown that tunicamycin inhibits the synthesis of such an intermediate. When isolated polysomes were placed into an in vitro glycosylating mixture containing Mn²⁺, GDP-[¹⁴C]mannose, and smooth membranes from A. niger nascent chains were labeled. This reaction was shown to be dependent on addition of polysomes to the mixture and was not inhibited by 2-deoxy-d-glucose or tunicamycin. Both in vivo and in vitro glycosylated nascent chains were found to have about the same size range, and so it is suggested that in vitro no new oligosaccharide chains were synthesized, but preexisting chains were extended.     

Oriciopsin,a new ring-D cleaved tetranortriterpenoid,and flindersiamine from Oriciopsis glaberrima

A novel ring-D cleaved tetranortriterpenoid, oriciopsin and flindersiamine have been isolated from the whole plant extracts of Oriciopsis glaberrima. The structure of the new limonoid was determined from its ¹H NMR and ¹³C NMR spectra and by mass spectrometry.     

A diterpenoid of the atis-16-ene class from Elaeoselinum foetidum

A new diterpenoid has been isolated from the root of Elaeoselinum foetidum. Its structure was established as ent-15α-senecioxy-atis-16-en-19-oic acid mainly by ¹H NMR ¹³C NMR spectroscopic studies of its methyl ester derivative.     

Sulcatine,a norsesquiterpene from the fungus Laurilia sulcata

The structure of sulcatine, a novel protoilludene norsesquiterpene isolated from a culture of Laurilia sulcata, has been assigned on the basis of a detailed study of its ¹H NMR and ¹³C NMR spectra and chemical evidence. The relative configuration was deduced from the observed ¹H {¹H} nuclear Overhauser effects (NOEs) and from the values of ¹H¹H coupling constants. The application of the exciton chirality method on the dibenzoate permitted establishment of the absolute configuration of sulcatine as 2S,3R,7S,8S,9R.     

2-β-d-Glucopyranosyloxy-2-methylpropanol in Acacia sieberana var. woodii

A new diol glucoside, 2-β-d-glucopyranosyloxy-2-methylpropanol, the first reported naturally occurring monoglucoside of an aliphatic dihydric alcohol, was isolated from pods of Acacia sieberana var. woodii. Structure elucidation was based on ¹ H and ¹³C NMR spectroscopy, and enzymatic analyses. The compound was hydrolysed very slowly by almond β-glucosidase, but cleaved by a β-glucuronidase enzyme complex from Helix pomatia.     

Methoxystyrenes from the genus Zieria

A new group of aromatic ethers has been isolated from the essential oils of several Zieria species. ¹H NMR and MS have indicated trimethoxy-, t