HIV-1 nucleocapsid protein zinc finger structures induce tRNA(Lys,3) structural changes but are not critical for primer/template annealing.

Retroviral reverse transcriptases use host cellular tRNAs as primers to initiate reverse transcription. In the case of human immunodeficiency virus type 1 (HIV-1), the 3' 18 nucleotides of human tRNA(Lys,3) are annealed to a complementary sequence on the RNA genome known as the primer binding site (PBS). The HIV-1 nucleocapsid protein (NC) facilitates this annealing. To understand the structural changes that are induced upon NC binding to the tRNA alone, we employed a chemical probing method using the lanthanide metal terbium. At low concentrations of NC, the strong terbium cleavage observed in the core region of the tRNA is significantly attenuated. Thus, NC binding first results in disruption of the tRNA's metal binding pockets, including those that stabilize the D-TPsiC tertiary interaction. When NC concentrations approach the amount needed for complete primer/template annealing, NC further destabilizes the tRNA acceptor-TPsiC stem minihelix, as evidenced by increased terbium cleavage in this domain. A mutant form of NC (SSHS NC), which lacks the zinc finger structures, is able to anneal tRNA(Lys,3) efficiently to the PBS, and to destabilize the tRNA tertiary core, albeit less effectively than wild-type NC. This mutant form of NC does not affect cleavage significantly in the helical regions, even when bound at high concentrations. These results, as well as experiments conducted in the presence of polyLys, suggest that in the absence of the zinc finger structures, NC acts as a polycation, neutralizing the highly negative phosphodiester backbone. The presence of an effective multivalent cationic peptide is sufficient for efficient tRNA primer annealing to the PBS.

Antifungal sterol biosynthesis inhibitors.

During the course of the last decade, the development of SBIs, and particularly sterol biomethylation inhibitors, has been based on the rational design approach. Successful though this approach has been in elucidating sterol biomethylation enzymology, its limitations are becoming apparent from the findings that: (i) 24,25-double bond metabolism gives rise to cholesterol and ergosterol in a mechanistically similar manner, (ii) 25-azasterols are harmful to human physiology, and (iii) side-chain modified sterols designed to inhibit the SMT enzyme in S. cerevisiae may be ineffective or operate by another kinetic mechanism in a related organism, rendering it therapeutically nonuseful. Nevertheless, it may be possible to ultimately capitalize on the unique aspects of sterol biomethylation chemistry and enzymology to design taxa-specific inhibitors. With increased understanding of the structure and function of SMT enzymes in different fungi, it should be possible to prepare novel mechanism-based inactivators to control SMT activity uniquely and with high specific activity.

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.

Characterization and catalytic properties of the sterol 14alpha-demethylase from Mycobacterium tuberculosis.

Sterol 14alpha-demethylase encoded by CYP51 is a mixed-function oxidase involved in sterol synthesis in eukaryotic organisms. Completion of the Mycobacterium tuberculosis genome project revealed that a protein having homology to mammalian 14alpha-demethylases might be present in this bacterium. Using genomic DNA from mycobacterial strain H(37)Rv, we have established unambiguously that the CYP51-like gene encodes a bacterial sterol 14alpha-demethylase. Expression of the M. tuberculosis CYP51 gene in Escherichia coli yields a P450, which, when purified to homogeneity, has the predicted molecular mass, ca. 50 kDa on SDS/PAGE, and binds both sterol substrates and azole inhibitors of P450 14alpha-demethylases. It catalyzes 14alpha-demethylation of lanosterol, 24, 25-dihydrolanosterol, and obtusifoliol to produce the 8,14-dienes stereoselectively as shown by GC/MS and (1)H NMR analysis. Both flavodoxin and ferredoxin redox systems are able to support this enzymatic activity. Structural requirements of a 14alpha-methyl group and Delta(8(9))-bond were established by comparing binding of pairs of sterol substrate that differed in a single molecular feature, e.g., cycloartenol paired with lanosterol. These substrate requirements are similar to those established for plant and animal P450 14alpha-demethylases. From the combination of results, the interrelationships of substrate functional groups within the active site show that oxidative portions of the sterol biosynthetic pathway are present in prokaryotes.

Structural requirements for substrate recognition of Mycobacterium tuberculosis 14 alpha-demethylase: implications for sterol biosynthesis.

Sterol 14 alpha-demethylase (14DM) is a cytochrome P-450 involved in sterol biosynthesis in eukaryotes. It was reported that Mycobacterium smegmatis also makes cholesterol and that cholesterol is essential to Mycobacterium tuberculosis (MT) infection, although the origin of the cholesterol is unknown. A protein product from MT having about 30% sequence identity with eukaryotic 14 alpha-demethylases has been found to convert sterols to their 14-demethyl products indicating that a sterol pathway might exist in MT. To determine the optimal sterol structure recognized by MT 14DM, binding of 28 sterol and sterol-like (triterpenoids) molecules to the purified recombinant 14 alpha-demethylase was examined. Like eukaryotic forms, a 3 beta-hydroxy group and a 14 alpha-methyl group are essential for substrate acceptability by the bacterial 14 alpha-demethylase. The high affinity binding of 31-norcycloartenol without detectable activity indicates that the Delta(8)-bond is required for activity but not for binding. As for plant 14 alpha-demethylases, 31-nor-sterols show a binding preference for MT 14DM. Similar to enzymes from mammals and yeast, a C24-alkyl group is not required for MT 14DM binding and activity, whereas it is for plant 14 alpha-demethylases.Thus, substrate binding to MT 14DM seems to share common features with all eukaryotic 14 alpha-demethylases, the MT form seemingly having the broadest substrate recognition of all forms of 14 alpha-demethylase studied so far. - Bellamine, A., A. T. Mangla, A. L. Dennis, W. D. Nes, and M. R. Waterman. Structural requirements for substrate recognition of Mycobacterium tuberculosis 14 alpha-demethylase: implications for sterol biosynthesis. J. Lipid Res. 2001. 42: 128;-136.