Evelyn A. Havir

l-Phenylalanine ammonia-lyase: IV. Evidence that the prosthetic group contains a dehydroalanyl residue and mechanism of action

Abstract l -Phenylalanine ammonia-lyase from potato tubers upon reduction with tritiated NaBH4 gave Type I tritiated enzyme. The nature of the reduction procedure, the observed correlation between loss of enzyme activity and tritium incorporation, and the magnitude of the incorporation suggested that reduction of an electrophilic center at the active site took place, and that one atom of hydrogen from borohydride is incorporated per active site reduced. Upon HCl hydrolysis, Type I tritiated enzyme gave exchangeable tritium and dl alanine-t in which most, but probably not all, of the tritium was in the β-position. It is proposed that the active site contains a dehydroalanyl residue which upon reduction yields a tritiated alanyl residue so substituted at the nitrogen atom that HCl hydrolysis leads to racemization and tritium loss. The unusual chemical environment of the alanyl residue is indicated by the mildness of the hydrolysis conditions necessary to release dl -alanine-β-t and the observation that Type I tritiated enzyme upon dilution and dialysis yielded exchangeable tritium and a new form of tritiated enzyme (Type II) which gave a neutral tritiated substance upon HCl hydrolysis. Guanidine hydrochloride also brings about the Type I to Type II conversion. The enzyme functions catalytically by converting the NH3+ of l -phenylalanine into a better leaving group. If the nitrogen of the dehydroalanyl residue is present as a Schiffs base masked against attack by such reagents as borohydride, then it is probable that the amino group of phenylalanine adds to the β-position of the dehydroalanyl double bond. If this intermediate undergoes a prototropic shift the conjugated system OCCαCβN may be formed (the three carbon atoms are those of the original dehydroalanyl residue and the nitrogen belongs to phenylalanine). With such a leaving group the free energy of the transition state in the elimination process should be lowered by resonance.

Oxalate production by virulent but not by hypovirulent strains of Endothia parasitica

Abstract Three virulent strains of Endothia parasitica when grown on potato dextrose agar containing methionine and biotin (PDAmb) or minimal media containing glucose supplemented with either glycolate or glyoxylate produced 10–12, 50–60 and 20–40 mg oxalate g−1 dry weight of fungus respectively. Oxalate accumulation was detected the fourth day after inoculation when the strains were grown on PDAmb or the glycolate medium but not until day 7 on the glyoxylate medium. In the early stages of growth (up to 0·1 g dry weight per colony) the total amount of oxalate excreted into the medium was proportional to the dry weight of the fungus but there was little or no correlation at later stages of growth. Oxalate was detected at the edge of the fungal colony, which is important if oxalate is to play a role in pathogenicity. Under identical conditions three hypovirulent strains, derived from the virulent ones by the introduction of cytoplasmic determinants for hypovirulence, produced no detectable oxalate.

A nonphotochemical-quenching-deficient mutant of Arabidopsis thaliana possessing normal pigment composition and xanthophyll-cycle activity.

Abstract. Higher-plant chloroplasts alter the distribution of absorbed radiant energy between photosynthesis and heat formation in response to changing illumination level or environmental stress. Fluorescence imaging was used to screen 62 yellow-green T-DNA insertion mutant lines of Arabidopsis thaliana (L.) Heynh. for reduced photoprotective nonphotochemical quenching (NPQ) capacity. Pulse-modulation fluorometry was employed to characterize one line (denoted Lsr1−) that exhibited an approximately 50% reduction in NPQ compared to the wild type (WT). The loss in NPQ capacity was associated with the ΔpH-dependent phase of quenching (qE). Under the growth conditions employed, pigment composition and levels of the six photosystem-II light-harvesting chlorophyll a/b proteins were identical in mutant and WT. Changes in the in-vivo levels of the xanthophyll pigments violaxanthin, antheraxanthin, and zeaxanthin in excess light were the same for mutant and WT. However, use of the violaxanthin de-epoxidase inhibitor dithiothreitol indicated that a zeaxanthin-dependent component of NPQ was specifically reduced in the mutant. The mutant exhibited diminished suppression of minimum fluorescence yield (Fo) in intense light suggesting an altered threshold in the mechanism of response to light stress in the mutant. The NPQ-deficient phenotype was meiotically transmissible as a semidominant trait and mapped near marker T27K12 on chromosome 1. The results suggest that the mutant is defective in sensing the transthylakoid ΔpH that reports exposure to excessive illumination.

Purification and properties of violaxanthin de-epoxidase from spinach

Abstract The enzyme violaxanthin de-epoxidase (VDE) catalyzes the conversion of violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z). It has been purified 194-fold with a yield of 1.4% from a sonicate of thylakoids of Spinacea oleracea to a specific activity of 19 μmol Z + A/min per mg protein. Purification steps included chromatography on DEAE-Sephadex, hydrophobic interaction chromatography on Butyl Sepharose, isoelectric-focusing, and gel filtration on Sephadex G-100. A single peptide band of 43.3 kDa was detected on SDS-PAGE, the apparent molecular mass of native enzyme was 45.8 kDa by gel filtration, and the pI was 4.95 on isoelectric focusing. The enzyme required ascorbate for activity, had a pH optimum of 5.2 and was stimulated three-fold by the addition of monogalactosyldi-acylglycerol (MGDG). The K m s for V and A were 5 and 5.3 μM, respectively. VDE was inhibited 50% by dithiothreitol (DTT), mercaptoethanol, cysteine and o -phenanthroline at 0.055, 0.68, 2.7 and 0.025 mM, respectively. With both DTT and o -phenanthroline, the rate of conversion of V to A was relatively unchanged whereas the conversion of A to Z was 50–80% inhibited. The enzyme also exhibited product inhibition by Z.

Modification of L-phenylalanine ammonia-lyase in soybean cell suspension cultures by 2-aminooxyacetate and L-2-aminooxy-3-phenylpropionate.

Suspension-cultured cells of soybean (Glycine max (L.) Merr. cv. Kanrich) produce large amounts of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), the first enzyme of phenylpropanoid metabolism, during growth. 2-Aminooxyacetic acid (AOA) and l-2-aminooxy-3-phenylpropionic acid (l-AOPP) inhibit the enzyme competitively in vitro and have been used for in vivo studies. The amount of extractable enzyme in the cells and their utilization of NO3−and NH3+are reduced upon the addition of AOA. When AOA was added at various times during growth, the appearance of additional enzyme activity was prevented but enzyme already formed was not inhibited. No evidence was obtained for the presence of an inhibitor in the extracts and AOA inhibition in vitro was readily reversible. It is conculded that AOA acts to inhibit the formation of PAL in suspension-cultured soy bean cells. In vitro inhibition of soybean PAL by l-AOPP could not be reversed; in contrast, the inhibition of maize (Zea mays L.) PAL was readily reversible. Added l-AOPP, which was rapidly taken up by the soybean cells, prevented the large increase in enzyme activity. Although PAL activity was blocked in the cultures, no appreciable increase in phenylalanine content could be detected in cell extracts. The response of soybean cell suspensions to l-AOPP addition thus differs from that of other tissues which in presence of l-AOPP show an increase in PAL activity and an accumulation of phenylalanine.

Purification and characterization of an isozyme of catalase with enhanced-peroxidatic activity from leaves of Nicotiana sylvestris

Two isozymes of catalase (EC 1.11.1.6), one with typically low peroxidatic activity (CAT-1) and the other with enhanced-peroxidatic activity (EP-CAT or CAT-3) have been purified to electrophoretic homogeneity from tobacco (Nicotiana sylvestris) seedlings and antibodies prepared against each. The isozyme proteins showed no immunological cross-reactivity. The subunit Mr was 55,300 +/- 750 for CAT-1 and 53,300 +/- 850 for CAT-3 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the catalatic reaction, the apparent Km values for CAT-1 and CAT-3 were 0.057 and 0.054 M, respectively, and the kcat values were 4.8 x 10(7) and 3.0 x 10(6) min-1, respectively. In the peroxidatic reaction, both have similar apparent Kms for H2O2. The apparent Km values for CAT-3 for the series methyl, ethyl, propyl, butyl, and allyl alcohols were 2.48, 5.6, 38.6, 429, and 16.3 mM, respectively. For CAT-1, the values were 697, 55.8, no detectable reaction with propyl and butyl, and 163 mM, respectively. Neither isozyme utilized dianisidine or guaiacol in the peroxidatic reaction. Catalase activity (CAT-2) which eluted in an intermediate position between CAT-1 and CAT-3 from a chromatofocusing column was composed of only one subunit whose Mr coincided with CAT-1, and only the antibody to CAT-1 reacted with CAT-2 protein. Thus, CAT-2 and CAT-1 appear closely related while CAT-3 is distinctly different.

An Introduction to the Enzymology of Phenylpropanoid Biosynthesis

It is the first purpose of this review to provide an outline of phenylpropanoid biosynthesis that can serve as an introduction to the work described in the next few chapters. The enzymology of three early steps in the metabolic pathway will be considred in some detail. These short reviews both point to recent findings and exemplify the progress, difficulties, and goals of the field. One purpose for studying the enzymology of such steps in detail is to make it possible for the plant physiologist to investigate the living plant in a rational and productive way. An enormous amount of painstaking and excellent work has already been carried out on the effects of light, hormones, temperature, infection, and development on various aspects of phenylpropanoid metabolism.11 8 99 100No attempt will be made to review this material, which falls within the scope of a later chapter, but questions of relevance to physiological studies will be discussed where they are suggested by the enzymology.

Oxaloacetate acetylhydrolase activity in virulent and hypovirulent strains of Endothia (Cryphonectria) parasitica

Abstract Extracts of three virulent strains of Endothia (Cryphonectria) parasitica and three hypovirulent strains derived from them were assayed for enzyme activities that would produce oxalate. Oxaloacetate acetylhydrolase (E.C. 3.7.1.1), the enzyme that converts oxaloacetate to oxalate and acetate, was at least four times higher in extracts of the virulent strains than in extracts of the hypovirulent strains. The level of activity of this enzyme could account for the formation of all the oxalate produced by the virulent cultures. When extracts of hypovirulent strains were added to extracts of virulent strains, the measured rate was less than the sum of the two rates. The inhibition did not change when the hypovirulent extract was either dialysed or boiled. No enzyme activity for the conversion ofglycolate or glyoxylate to oxalate could be detected in any of the extracts. There was no apparent difference between the virulent and hypovirulent strains of the fungus on the basis of intracellular organic acid content except that fumarate concentration was lower in extracts of hypovirulent strains. The amounts of oxalate produced by the virulent strains when grown on solid medium with malate (an immediate precursor of oxaloacetate) and fumarate were much higher than when they were grown on solid medium with glycolate. Two of the hypovirulent strains produced no oxalate on any of the substrates. However, the hypovirulent strain which had the highest oxaloacetate acetylhydrolase activity produced oxalate cultured on malate and fumarate but none on glycolate.

A mutant of Nicotiana sylvestris deficient in serine glyoxylate aminotransferase activity : Callus induction and photorespiratory toxicity in regenerated plants.

SummaryA photorespiration mutant of Nicotiana sylvestris lacking serine: glyoxylate aminotransferase activity was isolated in the M2 generation following EMS mutagenesis. Mutants showing chlorosis in air and normal growth in 1% CO2 were fed [14C]-2-glycolate to examine the distribution of 14C among photorespiratory intermediates. Mutant strain NS 349 displayed a 9-fold increase in serine accumulation relative to wild-type controls. Enzyme assays revealed an absence of serine: glyoxylate aminotransferase (SGAT) activity in NS 349, whereas other peroxisomal enzymes were recovered at normal levels. Heterozygous siblings of NS 349 segregating air-sensitive M3 progeny in a 3∶1 ratio were shown to contain one half the normal level of SGAT activity, indicating that air sensitivity in NS 349 results from a single nuclear recessive mutation eliminating SGAT activity. Since toxicity of the mutation depends on photorespiratory activity, callus cultures of the mutant were initiated and maintained under conditions suppressing the formation of functional plastids. Plantlets regenerated from mutant callus were shown to retain the SGAT deficiency and conditional lethality in air. The utility of photorespiration mutants of tobacco as vehicles for genetic manipulation of ribulose bisphosphate carboxylase/oxygenase at the somatic cell level is discussed.

Phenylalanine ammonia-lyase: Purification and characterization from soybean cell suspension cultures

Abstract Soybean cell suspension cultures ( Glycine max L. cv. Kanrich) grown on high-nitrogen medium produce 50 mU/g fresh wt of phenylalanine ammonia-lyase [EC 4.1.3.5] 7–9 days after inoculation. Nitrate was not limiting when the peak of enzyme activity was reached. Phenylalanine ammonia-lyase was purified 53-fold to essentially electrophoretic homogeneity from cell extracts with 10% recovery. The enzyme was stable in crude extracts and through most stages of purification. No activity could be detected with tyrosine as substrate in either crude extracts or purified enzyme. The electrophoretic mobility was somewhat less than that of the enzyme from maize but both eluted from an agarose column at the same position and the molecular weight of the subunit was similar for both enzymes. Thus the soybean enzyme is composed of four subunits and the native enzyme is ~330,000 M r . The variation in structure and/or size and availability of hydrophobic regions among phenylalanine ammonia-lyases from four sources (potato, maize, Rhodotorula glutinis , and soybean) was shown by the different elution patterns they exhibited on columns of ω-aminoalkyl agarose (agarose-C n -NH 2 , n = 0 to 8). The order of increasing hydrophobicity is soybean, potato, maize, R. glutinis . The soybean enzyme exhibited negative cooperativity before hydroxylapatite chromatography and positive cooperativity afterward. This is the first example of positive cooperativity observed for phenylalanine ammonia-lyase.