Biomedical subjects

P M Palenchar

Publications and source records attributed to P M Palenchar.

5 recordsLinked to original sources

PubMed2004

Adaptive combinatorial design to explore large experimental spaces: approach and validation.

Systems biology requires mathematical tools not only to analyse large genomic datasets, but also to explore large experimental spaces in a systematic yet economical way. We demonstrate that two-factor combinatorial design (CD), shown to be useful in software testing, can be used to design a small set of experiments that would allow biologists to explore larger experimental spaces. Further, the results of an initial set of experiments can be used to seed further 'Adaptive' CD experimental designs. As a proof of principle, we demonstrate the usefulness of this Adaptive CD approach by analysing data from the effects of six binary inputs on the regulation of genes in the N-assimilation pathway of Arabidopsis. This CD approach identified the more important regulatory signals previously discovered by traditional experiments using far fewer experiments, and also identified examples of input interactions previously unknown. Tests using simulated data show that Adaptive CD suffers from fewer false positives than traditional experimental designs in determining decisive inputs, and succeeds far more often than traditional or random experimental designs in determining when genes are regulated by input interactions. We conclude that Adaptive CD offers an economical framework for discovering dominant inputs and interactions that affect different aspects of genomic outputs and organismal responses.

Adaptation, Physiological↗

PubMed2001

The role of the cysteine residues of ThiI in the generation of 4-thiouridine in tRNA.

The enzyme ThiI is common to the biosynthetic pathways leading to both thiamin and 4-thiouridine in tRNA. We earlier noted the presence of a motif shared with sulfurtransferases, and we reported that the cysteine residue (Cys-456 of Escherichia coli ThiI) found in this motif is essential for activity (Palenchar, P. M., Buck, C. J., Cheng, H., Larson, T. J., and Mueller, E. G. (2000) J. Biol. Chem. 275, 8283-8286). In light of that finding and the report of the involvement of the protein IscS in the reaction (Kambampati, R., and Lauhon, C. T. (1999) Biochemistry 38, 16561-16568), we proposed two mechanisms for the sulfur transfer mediated by ThiI, and both suggested possible involvement of the thiol group of another cysteine residue in ThiI. We have now substituted each of the cysteine residues with alanine and characterized the effect on activity in vivo and in vitro. Cys-108 and Cys-202 were converted to alanine with no significant effect on ThiI activity, and C207A ThiI was only mildly impaired. Substitution of Cys-344, the only cysteine residue conserved among all sequenced ThiI, resulted in the loss of function in vivo and a 2700-fold reduction in activity measured in vitro. We also examined the possibility that ThiI contains an iron-sulfur cluster or disulfide bonds in the resting state, and we found no evidence to support the presence of either species. We propose that Cys-344 forms a disulfide bond with Cys-456 during turnover, and we present evidence that a disulfide bond can form between these two residues in native ThiI and that disulfide bonds do form in ThiI during turnover. We also discuss the relevance of these findings to the biosynthesis of thiamin and iron-sulfur clusters.

Alanine↗

PubMed2000

Evidence that ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis, may be a sulfurtransferase that proceeds through a persulfide intermediate.

ThiI is an enzyme common to the biosynthetic pathways leading to both thiamin and 4-thiouridine in tRNA. Comparison of the ThiI sequence with protein sequences in the data bases revealed that the Escherichia coli enzyme contains a C-terminal extension displaying sequence similarity to the sulfurtransferase rhodanese. Cys-456 of ThiI aligns with the active site cysteine residue of rhodanese that transiently forms a persulfide during catalysis. We investigated the functional importance of this sequence similarity and discovered that, like rhodanese, ThiI catalyzes the transfer of sulfur from thiosulfate to cyanide. Mutation of Cys-456 to alanine impairs this sulfurtransferase activity, and the C456A ThiI is incapable of supporting generation of 4-thiouridine in tRNA both in vitro and in vivo. We therefore conclude that Cys-456 of ThiI is critical for activity and propose that Cys-456 transiently forms a persulfide during catalysis. To accommodate this hypothesis, we propose a general mechanism for sulfur transfer in which the terminal sulfur of the persulfide first acts as a nucleophile and is then transferred as an equivalent of S(2-) rather than S(0).

Amino Acid Sequence↗

PubMed1999

Using genomic information to investigate the function of ThiI, an enzyme shared between thiamin and 4-thiouridine biosynthesis.

The gene thiI encodes a protein (ThiI) that plays a role in the transfer of sulfur from cysteine to both thiamin and 4-thiouridine, but the reaction catalyzed by ThiI remains undetermined. Based upon sequence alignments, ThiI shares a unique "P-loop" motif with the PPi synthetase family, four enzymes that catalyze adenylation and subsequent substitution of carbonyl oxygens. To test whether or not this motif is critical for ThiI function, the Asp in the motif was converted to Ala (D189A), and a screen for in vivo 4-thiouridine production revealed the altered enzyme to be inactive. Further scrutiny of sequence data and the crystal structures of two members of the PPi synthetase family implicated Lys321 in the proposed adenylation function of ThiI, and the critical nature of Lys321 has been demonstrated by site-directed mutagenesis and genetic screening. Our results, then, indicate that ThiI catalyzes the adenylation of a substrate at the expense of ATP, a narrowing of possible reactions that provides a strong new basis for deducing the early steps in the transfer of sulfur from cysteine to both thiamin and 4-thiouridine.

Amide Synthases↗

PubMed1998

Identification of a gene involved in the generation of 4-thiouridine in tRNA.

All organisms modify the bases of their RNA after transcription. Relatively little is known about the functions that these chemical alterations serve and, with very few exceptions, even less has been established regarding the enzymology involved. One modified base of known function is 4-thiouridine at position 8 of certain bacterial tRNAs, which serves as a photosensor for near-UV light. A gene involved in the conversion of uridine at position 8 into 4-thiouridine has been identified by genetic screening and its role in 4-thiouridine generation has been confirmed biochemically. This same gene, thiI , has recently been shown to play a role in thiamin biosynthesis. The purification and characteristics of the purified protein are also reported.

Animals↗