Mutations at amino acid positions 63, 189, and 396 of human immunodeficiency virus type 1 reverse transcriptase (RT) partially restore the DNA polymerase activity of a Trp229Tyr mutant RT.

Trp229 is part of the nonnucleoside reverse transcriptase inhibitor (NNRTI)-binding pocket of HIV-1 reverse transcriptase (RT). It is also an important constituent of the so-called "primer grip." Using a recombinant virus assay, we tried to obtain recombinant virus containing a Trp229Phe or a Trp229Tyr mutation in its RT. Previous studies already established the very low DNA polymerase activities of both the Trp229Phe and the Trp229Tyr mutant RT enzymes. We were able to obtain a Trp229Tyr but not a Trp229Phe mutant virus. However, in addition to the Trp229Tyr mutation this mutant virus also contained an Ile63Met, a Val189Ile, and a Glu396Gly mutation in its RT. When we evaluated the quadruple mutant virus for sensitivity/resistance against a variety of NNRTIs, no significant difference with the sensitivity/resistance profile of the single Trp229Tyr mutant RT enzyme could be observed. We found that the three additional mutations partly restored the low RNA- and DNA-dependent DNA polymerase activities of the Trp229Tyr mutant enzyme. Kinetic analysis revealed that both template/primer binding and dNTP incorporation are affected by the Trp229Tyr mutation. Our findings demonstrate that a mutation at position 229 is unlikely to occur under NNRTI drug pressure due to the poor catalytic activity of the singly mutated RT and the favorable drug sensitivity profile of the mutated enzyme/viruses in both the absence and the presence of the compensatory mutations. Therefore, amino acid position 229 may be regarded as an excellent amino acid target within the NNRTI pocket for rational drug design.

Characterization of a novel laccase produced by the wood-rotting fungus Phellinus ribis.

The white-rot fungus Phellinus ribis produced a single form of laccase, which was purified to apparent electrophoretic homogeneity from cultures induced with 2,5-xylidine. This protein was a dimer, consisting of two subunits of 76 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carbohydrate analysis revealed that the enzyme contained about 28% carbohydrate content. The laccase appeared to be different from other known laccases by the UV-visible absorption spectrum analysis. One enzyme molecule contained one copper, one manganese, and two zinc atoms. The laccase showed optimal activity at pH 4.0-6.0, 5.0, and 6.0 with 2,6-dimethoxyphenol, ABTS [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)], and syringaldazine, respectively. The enzyme preferably oxidized dimethoxyphenol and aromatic amine compounds. The stability of the laccase was low at acidic pH, whereas it showed high stability at neutral pH and mild temperature. The N-terminal amino acid sequence revealed a very low homology with other microbial laccases. With some substrates, the addition of manganese and H2O2 resulted in a remarkable increase in the oxidation rate. Without an appropriate phenolic substrate, the enzyme could not oxidize Mn(II) in the presence of H2O2 or pyrophosphate.

Pseudonocardia kongjuensis sp. nov., isolated from a gold mine cave.

The taxonomic position of an isolate that was recovered from a gold mine cave near Kongju, Republic of Korea, was determined by 16S rDNA sequence studies and chemotaxonomic characterization. Comparative studies of 16S rDNA sequences indicated that this organism was phylogenetically related to members of the genus Pseudonocardia, branching outside a cluster encompassing Pseudonocardia autotrophica and Pseudonocardia compacta. The affiliation to the genus was also supported by the cell chemistry, which was represented by a type IV cell wall, MK-8(H4) as the major menaquinone, a phospholipid type PIII pattern (phosphatidylcholine as a diagnostic phospholipid) and a DNA G+C content of 71 mol%. The fatty acid profile contained saturated, unsaturated and 10-methyl branched fatty acids, but tuberculostearic acid and hydroxy fatty acids were not present. The isolate differed from its phylogenetic neighbours in the presence of phosphatidylethanolamine, dodecanoate, 16-methylheptadecenoate and 16-methylheptadecanoate and the absence of phosphatidylinositol mannoside and phosphatidylmethylethanolamine. The unique combination of physiological properties, the cellular fatty acid profile and DNA-DNA hybridization data indicates that this organism is readily differentiated from the type strains of all of the validly published species of the genus Pseudonocardia. The name Pseudonocardia kongjuensis sp. nov. is proposed for the type strain, LM 157T (= IMSNU 50583T = KCTC 9990T = DSM 44525T).

Measurement of oxidized glutathione by enzymatic recycling coupled to bioluminescent detection.

A new method is described for the quantification of oxidized glutathione (GSSG) in tissues by enzymatic recycling coupled to NADPH bioluminescent detection. Tissue samples are treated with metaphosphoric acid. In a first step, after derivatization of GSH with 4-chloro-7-trifluoromethyl-1-methylquinolinium (CFQ), GSSG is recycled in the presence of dithionitrobenzoic acid (DTNB) and NADPH by glutathione reductase. In a second step, the GSSG-dependent NADPH consumption is measured by luminescence with NADPH:FMN oxidoreductase-bacterial luciferase. The coefficient of variation for GSSG measurements on repeated assays (n = 5) is 2 and 3% for standards and tissue samples, respectively. The sensitivity of this method is at the picomole level and is convenient for determination of GSSG physiological concentrations in tissues: GSSG levels measured in rat liver and kidney ranged from 76 to 215 and 52 to 170 nmol/g wet weight, respectively.

Creatine kinase isoenzymes specificities: histidine 65 in human CK-BB, a role in protein stability, not in catalysis.

Creatine kinases (CK) play a prominent role in cell energy distribution through an energy shuttle between mitochondria and other organelles. Human brain CK was cloned and overexpressed in COS-7 cells. We then deleted His-65 and/or Pro-66 situated near the center of a flexible loop as shown by X-ray crystallography on mitochondrial and cytosolic CK. The DeltaH65 mutant had nearly the same affinity for its substrates as wild isoenzyme, but its stability was very low. Unlike DeltaH65, DeltaH65P66 had a eightfold decreased affinity for creatine phosphate and was unable to dephosphorylate cyclocreatine phosphate. Our results demonstrate that, despite an overall similar shape of the proteins, this loop accounts for some subtle differences in isoenzyme functions.

Firefly luciferase generates two low-molecular-weight light-emitting species.

A bioluminescent D-luciferin-luciferase mixture is separated by gel filtration during the time course of the reaction. A simultaneous analysis with an UV-visible diode array detector and an on-line luminometer gives nonsuperimposable chromatograms. Luminescence recordings display three peaks, one associated with the enzyme (light-emitting species 1: LES(1)), and two other species free from the luciferase: LES(2), with a luciferyl-adenylate-like spectrum and LES(3). Production of these two species is nucleotide (ATP or 2'-dATP)- and pH-dependent. The chromatographic data presented here could lead to reconsideration of the generally assumed emission mechanism, which involves one emitter only. It could also suggest that each free emitting species is related to a colour of emission corresponding to the two defined wavelengths previously described ( approximately 575 and approximately 620 nm).

The emitting species dissociated from the enzyme can emit the light in Photinus pyralis luciferase system.

By fluorescent results, it has been proposed that the product (excited oxyluciferin) bounded in the firefly luciferase emits the light. Also it is generally accepted that two forms (keto and enol) of the oxyluciferin emit red and yellow-green light, respectively. In order to demonstrate the existence of free emitting species using His-tagged luciferase (His-luc), N-terminal 6x His-tagged luciferase (Photinus pyralis) was prepared and expressed in E. coli. After immobilization of His-luc on the membrane by IDA-Ni(2+)-His complex, His-luc clearly showed spectral changes of the emission toward red light. The luciferase-free product obtained from enzymatic reaction mixture in the presence of ATP and dATP emits the light with maximal wavelengths of 575 and 620 nm, respectively. Based on these results, we suppose that two different emitting species or two different energy levels of the emitting species are responsible for two different color lights.

Firefly luciferase has two nucleotide binding sites: effect of nucleoside monophosphate and CoA on the light-emission spectra.

A laboratory-made spectroluminometer was used to analyse the light emitted by firefly (Photinus pyralis) luciferase reacting with several nucleotide derivatives. The analysis of the light emission in the presence of ATP or dATP provides some evidence that the enzyme has two nucleotide binding sites, each one leading to the formation of a complex emitting mainly at 575 nm (ATP) or 610 nm (dATP). AMP is able to displace dATP from the second site (610 nm) to the first one. Photoaffinity labelling of the second site by 8-azido-AMP gives similar results. The amplification effect of CoA and acetyl-CoA is also reconsidered according to this model.

Identification of the creatine binding domain of creatine kinase by photoaffinity labeling.

A new photoaffinity probe with a benzophenone group, N-dibenzylphospho-N'-(4-benzoyl)-benzylguanidine (BzPG), has been synthesized on the basis on our previously described creatine kinase bisubstrate analog. BzPG is also a bisubstrate type analog whose photoinsertion is inhibited by the natural substrates of creatine kinase. When rabbit CK-MM is irradiated in the presence of BzPG then cleaved by CNBr, one labeled peptide can be purified by reverse phase HPLC and sequenced. This sequence of 31 amino acids (Ala30-Val60) contains a region which could be responsible for isoenzyme selectivity and another one just preceding the 11 amino acid peptide (Asp61-Thr70) very recently described as a putative creatine binding site. This second peptide was deduced from the comparison of 18 amino acid sequence alignments. We proposed the creatine binding site to be essentially a peptide from Lys39 to Val71.

N-dibenzylphospho-N'-3-(2,6-dichlorophenyl)propyl-guanidine is a bisubstrate-analog for creatine kinase.

We describe a new compound, N-dibenzylphospho-N'-3-(2,6-dichlorophenyl)-propylguanidine (DPPG), and our study of its interaction with cytosolic CK. To our knowledge, it is the most potent inhibitor known for CK: the Ki value versus ADP was 330 nM and 110 nM for CK-MM and BB respectively. In view of the inhibition pattern, Ki(app) dependencies on the second substrate, and very low Ki values, we conclude that DPPG binds to the active site as a bisubstrate-type analog. This bisubstrate analog confirms different mechanisms for CK-MM and BB: in spite of a more than 80% similarity in amino-acid sequences, both isoenzymes are random at pH 8.6 but CK-BB has an ordered mechanism at pH 6.6.

Dichloroaromatic phosphoguanidines are potent inhibitors but very poor substrates for cytosolic creatine kinase.

New phosphorylated guanidines have been synthesized and examined as potential inhibitors for creatine kinase. These compounds show a significant increase of inhibitory activity in comparison with the corresponding guanidines. Unlike the guanidines, they are competitive inhibitors because of the phosphoryl group. N-Phospho-N'-2-(2,6-dichlorophenyl)ethylguanidine is a potent inhibitor (K(i) = 2.0 mM and 1.2 mM respectively for muscle and brain-type creatine kinase). Although these phosphorylated analogs of creatine phosphate have a very poor substrate activity in the reverse reaction, the phosphoryl group is important for binding to the enzyme.

Synthesis and differential properties of creatine analogues as inhibitors for human creatine kinase isoenzymes.

Fourteen new creatine analogues, all with a guanidine function and either a polar or an apolar group instead of the creatine carboxylic function, were tested as potential inhibitors for human creatine kinase by kinetic analysis of their effects on the reaction rate. Only compounds bearing an apolar aromatic moiety, which was spaced from the guanidine function by at least two bonds, proved to have a significant inhibitory activity and showed a mixed-type inhibition similar to that of creatine. Among these compounds 2,6-dichlorobenzylguanidine (Ki = 5.6 mM and 39.8 mM for muscle-type and brain-type creatine kinases, respectively) and 3-(2,6-dichlorophenyl)propylguanidine (Ki = 15 mM and 4.5 mM) were the more potent inhibitors and showed a significant isoenzyme selectivity between muscle- and brain-type creatine kinases. Our results are in agreement with recent data that suggest the location of a hydrophobic pocket near the guanidine-binding domain of the enzyme. The observed selectivity in isoenzyme inhibition may be useful to study structural differences in catalytic centers.