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1.  Diversity of Archaeosine Synthesis in Crenarchaeota 
ACS Chemical Biology  2011;7(2):300-305.
Archaeosine (G+) is found at position 15 of many archaeal tRNAs. In Euryarchaeota, the G+ precursor, 7-cyano-7-deazaguanine (preQ0), is inserted into tRNA by tRNA-guanine transglycosylase (arcTGT) before conversion into G+ by ARChaeosine Synthase (ArcS). However, many Crenarchaeota known to harbor G+ lack ArcS homologs. Using comparative genomics approaches, two families that could functionally replace ArcS in these organisms were identified: 1) GAT-QueC, a two-domain family with an N-terminal glutamine amidotransferase class-II domain fused to a domain homologous to QueC, the enzyme that produces preQ0; 2) QueF-like, a family homologous to the bacterial enzyme catalyzing the reduction of preQ0 to 7-aminomethyl-7-deazaguanine. Here we show that these two protein families are able to catalyze the formation of G+ in a heterologous system. Structure and sequence comparisons of crenarchaeal and euryarchaeal arcTGTs suggest the crenarchaeal enzymes have broader substrate specificity. These results led to a new model for the synthesis and salvage of G+ in Crenarchaeota.
doi:10.1021/cb200361w
PMCID: PMC3289047  PMID: 22032275
2.  Functional Promiscuity of the COG0720 Family 
ACS Chemical Biology  2011;7(1):197-209.
The biosynthesis of GTP derived metabolites such as tetrahydrofolate (THF), biopterin (BH4), and the modified tRNA nucleosides queuosine (Q) and archaeosine (G+) relies on several enzymes of the Tunnel-fold superfamily. A subset of these proteins include the 6-pyruvoyl-tetrahydropterin (PTPS-II), PTPS-III, and PTPS-I homologs, all members of the COG0720 family, that have been previously shown to transform 7,8-dihydroneopterin triphosphate (H2NTP) into different products. PTPS-II catalyzes the formation of 6-pyruvoyltetrahydropterin in the BH4 pathway. PTPS-III catalyzes the formation of 6-hydroxylmethyl-7,8-dihydropterin in the THF pathway. PTPS-I catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin in the Q pathway. Genes of these three enzyme families are often misannotated as they are difficult to differentiate by sequence similarity alone. Using a combination of physical clustering, signature motif, and phylogenetic co-distribution analyses, in vivo complementation studies, and in vitro enzymatic assays, a complete reannotation of the COG0720 family was performed in prokaryotes. Notably, this work identified and experimentally validated dual function PTPS-I/III enzymes involved in both THF and Q biosynthesis. Both in vivo and in vitro analyses showed that the PTPS-I family could tolerate a translation of the active site cysteine and was inherently promiscuous, catalyzing different reactions on the same substrate, or the same reaction on different substrates. Finally, the analysis and experimental validation of several archaeal COG0720 members confirmed the role of PTPS-I in archaeosine biosynthesis, and resulted in the identification PTPS-III enzymes with variant signature sequences in Sulfolobus species. This study reveals an expanded versatility of the COG0720 family members and illustrates that for certain protein families, extensive comparative genomic analysis beyond homology is required to correctly predict function.
doi:10.1021/cb200329f
PMCID: PMC3262898  PMID: 21999246
Queuosine; archaeosine; tetrahydrofolate; biopterin; tRNA modification; riboflavin; 6-pyruvoyl-tetrahydropterin synthase
4.  Towards a Systems Approach in the Genetic Analysis of Archaea: Accelerating Mutant Construction and Phenotypic Analysis in Haloferax volcanii 
Archaea  2010;2010:426239.
With the availability of a genome sequence and increasingly sophisticated genetic tools, Haloferax volcanii is becoming a model for both Archaea and halophiles. In order for H. volcanii to reach a status equivalent to Escherichia coli, Bacillus subtilis, or Saccharomyces cerevisiae, a gene knockout collection needs to be constructed in order to identify the archaeal essential gene set and enable systematic phenotype screens. A streamlined gene-deletion protocol adapted for potential automation was implemented and used to generate 22 H. volcanii deletion strains and identify several potentially essential genes. These gene deletion mutants, generated in this and previous studies, were then analyzed in a high-throughput fashion to measure growth rates in different media and temperature conditions. We conclude that these high-throughput methods are suitable for a rapid investigation of an H. volcanii mutant library and suggest that they should form the basis of a larger genome-wide experiment.
doi:10.1155/2010/426239
PMCID: PMC3017900  PMID: 21234384
5.  A Gateway platform for functional genomics in Haloferax volcanii: deletion of three tRNA modification genes 
Archaea  2009;2(4):211-219.
In part due to the existence of simple methods for its cultivation and genetic manipulation, Haloferax volcanii is a major archaeal model organism. It is the only archaeon for which the whole set of post-transcriptionally modified tRNAs has been sequenced, allowing for an in silico prediction of all RNA modification genes present in the organism. One approach to check these predictions experimentally is via the construction of targeted gene deletion mutants. Toward this goal, an integrative “Gateway vector” that allows gene deletion in H. volcanii uracil auxotrophs was constructed. The vector was used to delete three predicted tRNA modification genes: HVO_2001 (encoding an archaeal transglycosyl tranferase or arcTGT), which is involved in archeosine biosynthesis; HVO_2348 (encoding a newly discovered GTP cyclohydrolase I), which catalyzes the first step common to archaeosine and folate biosynthesis; and HVO_2736 (encoding a member of the COG1444 family), which is involved in N4-acetylcytidine (ac4C) formation. Preliminary phenotypic analysis of the deletion mutants was conducted, and confirmed all three predictions.
PMCID: PMC2686393  PMID: 19478918
Archaea; GTP-cyclohydrolase I; halophile; tRNA-modification
6.  Comparative Genomics Guided Discovery of Two Missing Archaeal Enzyme Families Involved in the Biosynthesis of the Pterin Moiety of Tetrahydromethanopterin and Tetrahydrofolate 
ACS Chemical Biology  2012;7(11):1807-1816.
C-1 carriers are essential cofactors in all domains of life, and in Archaea, these can be derivatives of tetrahydromethanopterin (H4-MPT) or tetrahydrofolate (H4-folate). Their synthesis requires 6-hydroxymethyl-7,8-dihydropterin diphosphate (6-HMDP) as the precursor, but the nature of pathways that lead to its formation were unknown until the recent discovery of the GTP cyclohydrolase IB/MptA family that catalyzes the first step, the conversion of GTP to dihydroneopterin 2′,3′-cyclic phosphate or 7,8-dihydroneopterin triphosphate [El Yacoubi, B.; et al. (2006) J. Biol. Chem., 281, 37586–37593 and Grochowski, L. L.; et al. (2007) Biochemistry46, 6658–6667]. Using a combination of comparative genomics analyses, heterologous complementation tests, and in vitro assays, we show that the archaeal protein families COG2098 and COG1634 specify two of the missing 6-HMDP synthesis enzymes. Members of the COG2098 family catalyze the formation of 6-hydroxymethyl-7,8-dihydropterin from 7,8-dihydroneopterin, while members of the COG1634 family catalyze the formation of 6-HMDP from 6-hydroxymethyl-7,8-dihydropterin. The discovery of these missing genes solves a long-standing mystery and provides novel examples of convergent evolutions where proteins of dissimilar architectures perform the same biochemical function.
doi:10.1021/cb300342u
PMCID: PMC3500442  PMID: 22931285
7.  Comparative Genomics Guided Discovery of Two Missing Archaeal Enzyme Families Involved in the Biosynthesis of the Pterin Moiety of Tetrahydromethanopterin and Tetrahydrofolate 
ACS Chemical Biology  2012;7(11):1807-1816.
C-1 carriers are essential cofactors in all domains of life, and in Archaea, these can be derivatives of tetrahydromethanopterin (H4-MPT) or tetrahydrofolate (H4-folate). Their synthesis requires 6-hydroxymethyl-7,8-dihydropterin diphosphate (6-HMDP) as the precursor, but the nature of pathways that lead to its formation were unknown until the recent discovery of the GTP cyclohydrolase IB/MptA family that catalyzes the first step, the conversion of GTP to dihydroneopterin 2′,3′-cyclic phosphate or 7,8-dihydroneopterin triphosphate [El Yacoubi, B.; et al. (2006) J. Biol. Chem., 281, 37586–37593 and Grochowski, L. L.; et al. (2007) Biochemistry46, 6658–6667]. Using a combination of comparative genomics analyses, heterologous complementation tests, and in vitro assays, we show that the archaeal protein families COG2098 and COG1634 specify two of the missing 6-HMDP synthesis enzymes. Members of the COG2098 family catalyze the formation of 6-hydroxymethyl-7,8-dihydropterin from 7,8-dihydroneopterin, while members of the COG1634 family catalyze the formation of 6-HMDP from 6-hydroxymethyl-7,8-dihydropterin. The discovery of these missing genes solves a long-standing mystery and provides novel examples of convergent evolutions where proteins of dissimilar architectures perform the same biochemical function.
doi:10.1021/cb300342u
PMCID: PMC3500442  PMID: 22931285

Results 1-7 (7)