Hydroxynitrile lyases: Functions and properties

Plant hydroxynitrile lyases (Hnl) have attracted the attention of bioorganic scientists for more than 90 years. However, the most important increase in knowledge of this class of enzymes has only arisen in the recent decade. The industrial application of these enzymes as biocatalysts for the synthesis of enantiomerically pure α-cyanohydrins may be responsible for the growing interest in this area.
The Hnls are involved in the catabolic degradation of cyanogenic glycosides, releasing HCN which serves as defense agent against herbivores and microbial attack, or as a nitrogen source. Hydroxynitrile lyases from various plant families appear to represent a new example of enzymes that originated from the convergent evolution of different precursor proteins. The enzymes have been classified into non-FAD- and FAD-containing proteins. FAD-containing enzymes have been isolated exclusively from the Rosaceae, whereas the FAD-independent Hnls, which are more heterogenous in structure, have been characterized from various plant families (Poaceae, Euphorbiaceae, Linaceae, Olacaceae. Filitaceae). The aim of this review is to present a general survey of the natural function and localization of this class of enzymes and a comprehensive summary of the biochemical and genetic data of the isolated proteins.     

Ammonia fungi of iriomote Island in the southern Ryukyus,Japan and a new ammonia fungus,Hebeloma luchuense

Species composition and fruiting season of ammonia fungi were investigated in Iriomote Island of the southern Ryukyus, Okinawa Prefecture, Japan.Castanopsis andPinus forests were surveyed and 10 species of ammonia fungi were collected, including one new agaric species,Hebeloma luchuense sp. nov. This new fungus is characterized by having a rooting, squamulose-scaly stipe and cortinate veil and forms ectomycorrhizae withCastanopsis cuspidata var.sieboldii. Although the general mushroom season in theCastanopsis forest in Iriomote island was very short and restricted to summer, ammonia fungi were observed to fruit throughout the year in urea-treated plots.     

Changes in organic matter composition of forest soil treated with a large amount of urea to promote ammonia fungi and the abilities of these fungi to decompose organic matter

Organic matter composition (lignin, holocellulose, 50% (v/v) methanol extract, water-soluble carbohydrate (WSC) and phenolics (WSP), petroleum ether extract, and ash) of A₀ layer soil treated with 700 g/m² of urea to promote ammonia fungi was investigated in a Japanese red pine (Pinus densiflora) forest. Nine species of fungi were found during the study period of 18 months after the treatment. Of these, seven species belong to the ammonia fungi. WSC content of the treated soil was lower than that of the control. Methanol extract content increased initially after the treatment, then decreased to below the control level. There were no consistent differences in other components between the treated plot and the control. The abilities to decompose cellulose, lignin, chitin, protein and lipid in 18 strains in 10 species of the ammonia fungi were also screened. Cellulose was not lysed byPseudombrophila deerata, Hebeloma spp. andLaccaria bicolor. Strong lignolytic activity was shown byLyophyllum tylicolor, Coprinus echinosporus andP. deerata. Chitin was decomposed byAmblyosporium botrytis, L. tylicolor, C. echinosporus andHebeloma vinosophyllum. All strains possessed proteolytic and lipolytic activities. Supply of glucose to the culture media resulted in weaker enzyme activities except for lignolytic ability.     

The influence of ammonia on the growth and photosynthesis of Ruppia drepanensis Tineo from Doñana National Park (SW Spain)

In a laboratory experiment, Ruppia drepanensis Tineo seedlings from a brackish marsh in Southern Spain were grown at 20 and 30 °C, at three different nitrogen levels. These levels were obtained by the addition of a slow release fertilizer (23% NH₄NO₃ by weight) to a sediment mixture of sand and clay (3:1). Several morphometric parameters were recorded during the first five weeks of the experiment, and photosynthesis and respiration were measured after 7 weeks of growth. Results showed a significant reduction of growth and development with increasing nitrogen and temperature levels. Dark respiration increased strongly at high nitrogen levels. At the same time, net photosynthesis at 250 and 500 µE m^-2 s^-1, Pm, Km and LCP were not affected by either factor. We attribute these phenomena to ammonia toxicity, since relatively high total ammonia (NH₃ + NH_f4 ^p+) levels were found in the interstitial water.     

The biosynthesis of rosmarinic acid in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei accumulate very high amounts of rosmarinic acid, an ester of caffeic acid and 3,4-dihydroxyphenyllactate, in medium with elevated sucrose concentrations. Since the synthesis of this high level of rosmarinic acid occurs in only five days of the culture period, the activities of the enzymes involved in the biosynthesis are very high. Therefore all the enzymes necessary for the formation of rosmarinic acid from the precursors phenylalanine and tyrosine could be isolated from cell cultures of Coleus blumei: phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, hydroxycinnamoyl:CoA ligase, tyrosine aminotransferase, hydroxyphenylpyruvate reductase, rosmarinic acid synthase and two microsomal 3- and 3-hydroxylases. The main characteristics of these enzymes of the proposed biosynthetic pathway of rosmarinic acid will be described.Abbreviations DHPL 3,4-dihydroxyphenyllactate - DHPP 3,4-dihydroxyphenylpyruvate - pHPL 4-hydroxyphenyllactate - pHPP 4-hydroxyphenylpyruvate - RA rosmarinic acid     

Effect of culture conditions on the isocitrate dehydrogenase and isocitrate lyase activities in Chlamydomonas reinhardtii

In the green alga Chlamydomonas reinhardtii , nitrogen staravation induced a reversible increase (2-fold) in NAD-isocitrate dehydrogenase (NAD-IDH; EC 1.1.1.41) and NADP-isocitrate dehydrogenase (NADP-IDH; EC 1.1.1.42) activities. Both enzymes were not affected by the concentration of CO₂, the dark or the nature of the nitrogen source (nitrate, nitrite, or ammonium). When cells growing autotrophically were transferred to heterotrophic conditions, a 40% reduction of the NAD-IDH activity was detected, a 2-fold increase of NADP-IDH was observed and isocitrate lyase (ICL; EC 4.1.3.1) activity was induced. The replacement of autotrophic conditions led to the initial activity levels. NAD- and NADP-IDH activities showed markedly different patterns of increase in synchronous cultures of this alga obtained by 12 h light/12 h dark transitions. While NAD-IDH increased in the last 4 h of the dark period, NADP-IDH increased during the last 4 h of the light period, remaining constant for the rest of the cycle.     

Field investigations of ammonia exchange between barley plants and the atmosphere. I. Concentration profiles and flux densities of ammonia

The exchange of ammonia between the atmosphere and the canopy of spring barley crops growing at three levels of nitrogen application (medium N, high N and excessive N) was studied over two consecutive growing seasons by use of micrometeorological techniques. In most cases, ammonia was emitted from the canopy to the atmosphere. The emission started around 2 weeks before anthesis, and peaked about or shortly after anthesis. The volatilization of ammonia only took place in the daytime. During the night-time, atmospheric ammonia was frequently aborbed by the canopy. Occasionally, plants in the medium and high N treatments also absorbed ammonia from the atmosphere during the daytime. Daytime absorption of ammonia never occurred in the excessive N canopy. The loss of ammonia from the canopy amounted in both years to 0.5–1.5 kg NH₃-N ha^?1 and increased with the N status of the canopy. In agreement with the small losses of ammonia, the content of ¹⁵N-labelled nitrogen in the plants did not decline during the grain-filling period. The experimental years were characterized by very favourable conditions for grain dry matter formation, and for re-utilization of nitrogen mobilized from leaves and stems. Consequently, a very high part of the nitrogen in the mature plants was located in grain dry matter (80–84% in 1989; 74–80% in 1990). The efficient re-utilization of nitrogen may have reduced the volatilization of ammonia.     

Genes of the R-phycocyanin II locus of marine Synechococcus spp., and comparison of protein-chromophore interactions in phycocyanins differing in bilin composition

R-phycocyanin II (RPCII) is a recently discovered member of the phycocyanin family of photosynthetic light-harvesting proteins. Genes encoding the and subunits of RPCII were cloned and sequenced from marine Synechococcus sp. strains WH8020 and WH8103. The deduced amino acid sequences of RPCII were compared to two other types of phycocyanin, C-phycocyanin (CPC) and phycoerythrocyanin (PEC). These three types vary in the composition of their covalently bound bilin prosthetic groups. In terms of amino acid sequence identity RPCII is highly homologous to CPC and PEC, suggesting that the known three-dimensional structures of the latter two are representative of RPCII. Thus the amino acid residues contacting the three bilins of RPCII could be inferred and compared to those in CPC and PEC. Certain residues were identified among the three phycocyanins as possibly correlating with specific bilin isomers. In overall sequence RPCII and CPC are more homologous to one another than either is to PEC. This probably reflects functional homology in the roles of RPCII and CPC in the transfer of light energy to the core of the phycobilisome, a function not attributed to PEC. The genomes of Synechococcus sp. strains WH8020, WH8103 and WH7803 share homologous open reading frames in the vicinity of RPCII genes. The nucleotide sequence extending 3 from RPCII genes in strain WH8020 revealed two open reading frames homologous to components of an CPC phycocyanobilin lyase. These open reading frames may encode a lyase specific for the attachment of phycoerythrobilin to RPCII.