Induction of oxidative stress in Prototheca zopfii by indole-3-acetic acid/HRP or 2,4-pentanedione/HRP systems and their oxidation products.

We investigated the toxic effects on Prototheca zopfii of indole-3-acetic acid (IAA) and 2,4-pentanedione (PD) combined with horseradish peroxidase (HRP) alongside the oxidation products of 3-methyl-2-oxindole (MOI) and indole-3-carbinol (I3C) from the IAA/HRP system and methylglyoxal (MGO) from the PD/HRP system. The microorganism was incubated in the absence (control) or presence of IAA, PD, IAA/HRP, PD/HRP, MOI, I3C and MGO and determined: (1) cytotoxicity by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay; (2) growth inhibitory concentration by resazurin assay and (3) antioxidant enzymes activities of: catalase (CAT), glutathione reductase (GR) and superoxide dismutase (SOD). P. zopfii was more susceptible to IAA at 40 mM than PD at the same concentration, which seems to indicate that IAA was more effective at initiating cell death. These data corroborate results from the resazurin assay. Concentrations of 40 mM of IAA, IAA/HRP and PD/HRP, 20 mM of PD/HRP, 10 mM of MOI, 2 mM of I3C and 8 mM of MGO inhibited the growth of P. zopfii. With sub-inhibitory concentrations of IAA and IAA/HRP at 30 mM, MOI at 8 mM and I3C at 1 mM, the activities of CAT and GR increased, whereas no statistical difference was observed for CAT activity with IAA/HRP. Thus, PD at 30 mM and MGO at 6 mM increased the activities of CAT and GR, whereas PD/HRP system at 15 mM decreased CAT activity and PD/HRP and MGO showed no statistical difference for SOD activity. In conclusion, IAA/HRP or PD/HRP systems and their oxidation products exert cytotoxic effects on P. zopffi; however, I3C and MGO appear to exert greater microbicidal effect on P. zopfii.

The pathway of L-ascorbic acid biosynthesis in the colourless microalga Prototheca moriformis.

When mutant strain UV77-247 of Prototheca moriformis Kruger was fed d-[1-13C]Glc, it synthesized l-ascorbic acid (AA) with approximately three-quarters of the label at the C-1 position and the remaining label at the C-6 position, showing that AA is made by a non-inversion (retention) pathway, i.e. C-1 of Glc becomes C-1 of AA. The label present at C-6 is consistent with the glycolytic conversion of Glc to 3-carbon intermediates and subsequent gluconeogenesis. Compounds suggested as intermediates in inversion-type pathways were not converted to AA. Most strains converted Man to AA at a rate greater than they did Glc. Enzyme activities leading from Fru-6-P to the formation of GDP-Man were identified in all strains, but none of these activities correlated with the mutants' abilities to accumulate AA. However, there was a strong correlation between GDP-Man-3,5-epimerase activity and AA accumulation. Wild-type P. moriformis ATCC 75669 and mutant strains of varying AA-synthesizing abilities rapidly converted l-Gal or l-galactono-1,4-lactone to AA. Based on this data, a biosynthetic pathway from Glc to AA is proposed in which the epimerase is the rate-limiting activity in AA synthesis.

The genes encoding subunits of ATP synthase are conserved in the reduced plastid genome of the heterotrophic alga Prototheca wickerhamii.

The heterotrophic unicellular alga Prototheca wickerhamii is closely related to the photoautotrophic Chlorella vulgaris but has a 54,100-bp plastid DNA (ptDNA) that is much smaller than the chloroplast DNA of C. vulgaris (150,613 bp). The nucleotide sequence of 28,093 bp of the Prototheca ptDNA has been determined. No genes for photosynthetic functions have been found, except for sequences encoding six subunits of the ATP synthase ( atpA, atpB, atpE, atpF, atpH, and atpI). Transcripts of these atp genes have also been detected. Whether the leucoplasts of Prototheca contain a functional ATP synthase has still to be elucidated. Identified genes further include tufA, minD, cysT, and genes coding for three rRNAs, 22 tRNAs, and 12 ribosomal proteins. The results support the idea that, in the reduced plastid genome of Prototheca, genes coding for components of the plastid translational apparatus have been preferentially retained, and might be needed for the expression of the atp genes and some unassigned ORFs.

Sterol C-methyl transferase from Prototheca wickerhamii mechanism, sterol specificity and inhibition.

The membrane-bound sterol methyl transferase (SMT) enzyme from Prototheca wickerhamii, a non-photosynthetic, yeast-like alga, was found to C-methylate appropriate delta24(25)-sterol acceptor molecules to delta25(27)-24beta-methyl products stereoselectively. Incubation with pairs of substrates--[2H3-methyl]AdoMet and cycloartenol, and AdoMet and [27-(13)C]lanosterol--followed by 1H and 13C NMR analysis of the isotopically labeled products demonstrated the si-face (beta-face attack) mechanism of C-methylation and the regiospecificity of delta25(27)-double bond formation from the pro-Z methyl group (C27) on lanosterol. The enzyme has a substrate preference for a sterol with a 3beta-hydroxyl group, a planar nucleus and a side chain oriented into a 'right-handed' structure (20R-chirality) characteristic of the native substrate, cycloartenol. The apparent native molecular weight of the SMT was determined to be approximately 154,000, as measured by Superose 6 FPLC. A series of sterol analogues which contain heteroatoms substituted for C24 and C25 or related structural modifications, including steroidal alkaloids, havs been used to probe further the active site and mechanism of action of the SMT enzyme. Sterol side chains containing isoelectronic modifications of a positively charged moiety in the form of an ammonium group substituted for carbon at C25, C24, C23 or C22 are particularly potent non-competitive inhibitors (Ki for the most potent inhibitor tested, 25-azacycloartanol, was ca. 2 nM, four orders of magnitude less than the Km for cycloartenol of 28 microM), supporting the intermediacy of the 24-methyl C24(25)-carbenium ion intermediate. Ergosterol, but neither cholesterol nor sitosterol, was found to inhibit SMT activity (Ki = 80 microM). The combination of results suggests that the interrelationships of substrate functional groups within the active center of a delta24(25) to delta25(27) 24beta-methyl-SMT could be approximated thereby allowing the rational design of C-methylation inhibitors to be formulated and tested.

4,4,14 alpha-trimethyl 9 beta,19-cyclo-5 alpha-26-homocholesta-24,26-dien-3 beta-ol: a potent mechanism-based inactivator of delta 24(25)- to delta 25(27)-sterol methyl transferase.

The title compound (4A) was synthesized and tested as a mechanism-based inactivator of the sterol methyl transferase (SMT) enzyme from Prototheca wickerhamii. Using cycloartenol as substrate, 4A was found to exhibit time-dependent inactivation kinetics, generating a Ki value of 30 microM and Kinact value of 0.30 min-1.

Investigation of the beta-lactamase activity of Prototheca zopfii.

A total of 52 strains of Prototheca zopfii were studied with a series of beta-lactam compounds in order to test possible existence of beta-lactamase capable of inactivating these compounds. The method used was based on the iodimetric technique. Results showed the existence of beta-lactamase which worked in different degrees depending on the beta-lactam compounds.