Ciarán N. Cronin

Biochemical characterization of recombinant hepatitis C virus nonstructural protein 4B: evidence for ATP/GTP hydrolysis and adenylate kinase activity.

While nonstructural protein 4B (NS4B) from hepatitis C virus (HCV) is absolutely required for viral propagation, a full understanding of the enzymatic properties of this protein is lacking. Previous studies suggest that NS4B is located at the endoplasmic reticulum and that the protein structure consists of four central transmembrane domains with the N- and C-termini located in the cytoplasm of the host cell. To characterize the enzymatic activity of NS4B, the full-length protein with a C-terminal His tag was expressed in Sf9 insect cells and stabilized with nonionic detergents during purification. Chemical cross-linking experiments using GTP-gamma-azidoanilide and ATP-gamma-azidoanilide and equilibrium binding analyses with GTPgammaS and ATPgammaS show that both GTP and ATP are bound by NS4B, with ATP displaying a higher affinity. Analyses of enzymatic reactions catalyzed by NS4B indicate that the terminal phosphate groups of ATP, GTP, and GDP are removed to produce ADP, GDP, and GMP, respectively. The k(cat) for hydrolysis of GTP by purified NS4B compared favorably with the k(cat) for hydrolysis of GTP by Ras-p21 in the absence of GTPase activating proteins (GAPs). In addition to the hydrolysis of NTP and NDP substrates, adenylate kinase activity was detected in purified preparations of NS4B with the reverse reaction 2ADP --> ATP + ADP, yielding a larger k(cat) compared to that of the forward reaction ATP + AMP --> 2ADP. These studies suggest that HCV NS4B possesses both adenylate kinase activity and nucleotide hydrolase activity. Mutation of amino acids in the Walker A and B motifs of NS4B resulted in decreased affinity for both GTPgammaS and ATPgammaS as well as decreased ATP hydrolysis and AK activity.

Organization and sequences of p-hydroxybenzaldehyde dehydrogenase and other plasmid-encoded genes for early enzymes of the p-cresol degradative pathway in Pseudomonas putida NCIMB 9866 and 9869.

The gene (designated pchA) encoding the aldehyde dehydrogenase that is required to metabolise the p-hydroxybenzaldehyde produced by the degradation of p-cresol in Pseudomonas putida NCIMB 9866 and 9869 has been identified on plasmids pRA4000 and pRA500, respectively. The gene lies immediately upstream of the pchC and pchF genes encoding the subunits of p-cresol methylhydroxylase (PCMH), the preceeding enzyme in the p-cresol degradative pathway. In pRA500 the latter genes are followed by the genes encoding the alpha (pcaG) and beta (pcaH) subunits of protocatechuate-3,4-dioxygenase, whereas in pRA4000 the genes encoding PCMH are followed by an open reading frame encoding a protein that is similar to the maturase-related protein of P. alcaligenes. A gene, designated pchX, that encodes a protein of unknown function was identified between the pchC and pchF genes in both plasmids.

Redesign of Choline Acetyltransferase Specificity by Protein Engineering

Since the development of site-directed mutagenesis techniques over 15 years ago (Zoller, M. J., and Smith, M. (1982) Nucleic Acids Res. 10, 6487–6500), it has been a goal of protein engineering to utilize the procedure to redesign existing enzyme structures to produce proteins with altered or novel catalytic properties. To date, however, the more successful achievements have relied exclusively on the availability of three-dimensional protein structure maps to direct the redesign strategies. Presently, such maps are unavailable for choline acetyltransferase and carnitine acetyltransferase, enzymes that catalyze the reversible transfer of an acetyl group from acetyl-CoA to choline and l-carnitine, respectively. A more empirical approach, based on cross-referencing substrate structure comparisons with protein alignment data, was used to redesign choline acetyltransferase to accommodate l-carnitine as an acceptor of the acetyl group. A mutant choline acetyltransferase that incorporates four amino acid substitutions from wild type, shows a substantial increase in catalytic efficiency (k cat/K m ) towardl-carnitine (1,620-fold) and shifts the catalytic discrimination between choline and l-carnitine by >390,000 in favor of the latter substrate. These dramatic alterations in catalytic function demonstrate that significant success in protein redesign can be achieved in the absence of three-dimensional protein structure data.