Robert Rodriguez

In contrast to all other chromosomally encoded dihydrofolate reductases characterized so far, dihydrofolate reductase (DHFR) from the hyperthermophile Thermotoga maritima forms a highly stable dimer. The dimer interface involves residues whose mobility is important for catalysis in monomeric DHFRs. Here, we report the generation of a variant of DHFR from T. maritima, TmDHFR-V11D, in which a single amino acid replacement was sufficient to favor the monomeric form of the enzyme in the presence of the nondenaturing zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The free energy of stabilization of monomeric TmDHFR-V11D was 15 kJ mol−1 lower than that of the wild-type dimer, while the melting temperature of monomeric TmDHFR-V11D was comparable to that of monomeric DHFR from the thermophile Bacillus stearothermophilus, supporting the hypothesis that oligomerization is required to achieve the thermal stabilities necessary for activity at temperatures optimal for growth of hyperthermophiles. Both the steady-state turnover numbers and rates of hydride transfer were reduced in TmDHFR-V11D. However, a similar reduction of the rate constants was observed in a different variant, TmDHFR-V126E, which remained as a dimer under all experimental conditions used here. Monomeric TmDHFR-V11D had a similar rate of hydride transfer to the dimeric form, but a reduced steady-state turnover rate. Intersubunit motions therefore appear to be less important than correlated motions within individual subunits for TmDHFR-catalyzed hydride transfer, but are critical to the overall progression of the catalytic cycle. Hence, the reduced catalytic activity of TmDHFR relative to the monomeric Escherichia coli enzyme is not caused by rigidity resulting from dimerization, but is a subtle consequence of the sequence and structure of its subunits, which appear to have evolved to allow thermostability at the expense of catalysis.

Dihydrofolate reductase (DHFR) maintains the intracellular pool of tetrahydrofolate through catalysis of hydrogen transfer from reduced nicotinamide adenine dinucleotide to 7,8-dihydrofolate. We report results for pre-steady-state kinetic studies of the temperature dependence of the rates and the hydrogen/deuterium-kinetic isotope effects for the reactions catalysed by the enzymes from the mesophilic Escherichia coli and the hyperthermophilic Thermatoga maritima. We propose an evolutionary pattern in which catalysis progressed from a relatively rigid active site structure in the ancient thermophilic DHFR to a more flexible and kinetically more efficient structure in E. coli that actively promotes hydrogen transfer at physiological pH by modulating the tunnelling distance. The E. coli enzyme appeared relatively robust, in that kinetically severely compromised mutants still actively propagated the reaction. The reduced hydrogen transfer rates of the extensively studied Gly121Val mutant of DHFR from E. coli were most likely due to sterically unfavourable long-range effects from the introduction of the bulky isopropyl group.

Synthesis of novel C-4 disubstituted beta-lactam that has N-methyl pyrrole system has been achieved through Staudinger cycloaddition reaction of acid chloride and imine. Interestingly, this reaction has produced a single stereoisomer.

Orangelo or best known as ‘Chironja’ is a hybrid citrus fruit between orange and grapefruit. Previous investigations agree that different compounds found in citrus fruits like orange and grapefruit inhibit, completely or partially, the activity of different isoenzymes in the cytochrome P450 system. These compounds are ascorbic acid, flavonoids, limonoids, furocoumarins and other related complexes. The effect of this hybrid in the P450 system of Chinese Hamster Ovarian Cells, especially in CYP3A4 isoenzyme was studied. Extraction from the fruit was performed by squeezing three chironjas. CHO cells were treated for 48 hours with the chironja's extract. After this procedure the luminescence of each sample was determined using an appropriate luminescent compound. Intensity was measured at 450 nm using a spectrofluorometer. Results were: 0.827 pmol d‐luciferin/ pmol/min., 2.44 pmol d‐luciferin/pmol/min., and 1.26 pmol d‐luciferin/pmol/min., control, organic extract and treated cells, respectively. An increase on enzyme activity was observed. Further studies are being conducted in order to establish the effect of this extract on the isoenzyme.