Comparative genomics of NAD biosynthesis in cyanobacteria.

Biosynthesis of NAD(P) cofactors is of special importance for cyanobacteria due to their role in photosynthesis and respiration. Despite significant progress in understanding NAD(P) biosynthetic machinery in some model organisms, relatively little is known about its implementation in cyanobacteria. We addressed this problem by a combination of comparative genome analysis with verification experiments in the model system of Synechocystis sp. strain PCC 6803. A detailed reconstruction of the NAD(P) metabolic subsystem using the SEED genomic platform (http://theseed.uchicago.edu/FIG/index.cgi) helped us accurately annotate respective genes in the entire set of 13 cyanobacterial species with completely sequenced genomes available at the time. Comparative analysis of operational variants implemented in this divergent group allowed us to elucidate both conserved (de novo and universal pathways) and variable (recycling and salvage pathways) aspects of this subsystem. Focused genetic and biochemical experiments confirmed several conjectures about the key aspects of this subsystem. (i) The product of the slr1691 gene, a homolog of Escherichia coli gene nadE containing an additional nitrilase-like N-terminal domain, is a NAD synthetase capable of utilizing glutamine as an amide donor in vitro. (ii) The product of the sll1916 gene, a homolog of E. coli gene nadD, is a nicotinic acid mononucleotide-preferring adenylyltransferase. This gene is essential for survival and cannot be compensated for by an alternative nicotinamide mononucleotide (NMN)-preferring adenylyltransferase (slr0787 gene). (iii) The product of the slr0788 gene is a nicotinamide-preferring phosphoribosyltransferase involved in the first step of the two-step non-deamidating utilization of nicotinamide (NMN shunt). (iv) The physiological role of this pathway encoded by a conserved gene cluster, slr0787-slr0788, is likely in the recycling of endogenously generated nicotinamide, as supported by the inability of this organism to utilize exogenously provided niacin. Positional clustering and the co-occurrence profile of the respective genes across a diverse collection of cellular organisms provide evidence of horizontal transfer events in the evolutionary history of this pathway.

Aerobic tryptophan degradation pathway in bacteria: novel kynurenine formamidase.

While a variety of chemical transformations related to the aerobic degradation of L-tryptophan (kynurenine pathway), and most of the genes and corresponding enzymes involved therein have been predominantly characterized in eukaryotes, relatively little was known about this pathway in bacteria. Using genome comparative analysis techniques we have predicted the existence of the three-step pathway of aerobic L-tryptophan degradation to anthranilate (anthranilate pathway) in several bacteria. Based on the chromosomal gene clustering analysis, we have identified a previously unknown gene encoding for kynurenine formamidase (EC 3.5.1.19) involved with the second step of the anthranilate pathway. This functional prediction was experimentally verified by cloning, expression and enzymatic characterization of recombinant kynurenine formamidase orthologs from Bacillus cereus, Pseudomonas aeruginosa and Ralstonia metallidurans. Experimental verification of the inferred anthranilate pathway was achieved by functional expression in Escherichia coli of the R. metallidurans putative kynBAU operon encoding three required enzymes: tryptophan 2,3-dioxygenase (gene kynA), kynurenine formamidase (gene kynB), and kynureninase (gene kynU). Our data provide the first experimental evidence of the connection between these genes (only one of which, kynU, was previously characterized) and L-tryptophan aerobic degradation pathway in bacteria.

Ribosylnicotinamide kinase domain of NadR protein: identification and implications in NAD biosynthesis.

NAD is an indispensable redox cofactor in all organisms. Most of the genes required for NAD biosynthesis in various species are known. Ribosylnicotinamide kinase (RNK) was among the few unknown (missing) genes involved with NAD salvage and recycling pathways. Using a comparative genome analysis involving reconstruction of NAD metabolism from genomic data, we predicted and experimentally verified that bacterial RNK is encoded within the 3' region of the nadR gene. Based on these results and previous data, the full-size multifunctional NadR protein (as in Escherichia coli) is composed of (i) an N-terminal DNA-binding domain involved in the transcriptional regulation of NAD biosynthesis, (ii) a central nicotinamide mononucleotide adenylyltransferase (NMNAT) domain, and (iii) a C-terminal RNK domain. The RNK and NMNAT enzymatic activities of recombinant NadR proteins from Salmonella enterica serovar Typhimurium and Haemophilus influenzae were quantitatively characterized. We propose a model for the complete salvage pathway from exogenous N-ribosylnicotinamide to NAD which involves the concerted action of the PnuC transporter and NRK, followed by the NMNAT activity of the NadR protein. Both the pnuC and nadR genes were proven to be essential for the growth and survival of H. influenzae, thus implicating them as potential narrow-spectrum drug targets.

From genetic footprinting to antimicrobial drug targets: examples in cofactor biosynthetic pathways.

Novel drug targets are required in order to design new defenses against antibiotic-resistant pathogens. Comparative genomics provides new opportunities for finding optimal targets among previously unexplored cellular functions, based on an understanding of related biological processes in bacterial pathogens and their hosts. We describe an integrated approach to identification and prioritization of broad-spectrum drug targets. Our strategy is based on genetic footprinting in Escherichia coli followed by metabolic context analysis of essential gene orthologs in various species. Genes required for viability of E. coli in rich medium were identified on a whole-genome scale using the genetic footprinting technique. Potential target pathways were deduced from these data and compared with a panel of representative bacterial pathogens by using metabolic reconstructions from genomic data. Conserved and indispensable functions revealed by this analysis potentially represent broad-spectrum antibacterial targets. Further target prioritization involves comparison of the corresponding pathways and individual functions between pathogens and the human host. The most promising targets are validated by direct knockouts in model pathogens. The efficacy of this approach is illustrated using examples from metabolism of adenylate cofactors NAD(P), coenzyme A, and flavin adenine dinucleotide. Several drug targets within these pathways, including three distantly related adenylyltransferases (orthologs of the E. coli genes nadD, coaD, and ribF), are discussed in detail.

Complete reconstitution of the human coenzyme A biosynthetic pathway via comparative genomics.

The biosynthesis of CoA from pantothenic acid (vitamin B5) is an essential universal pathway in prokaryotes and eukaryotes. The CoA biosynthetic genes in bacteria have all recently been identified, but their counterparts in humans and other eukaryotes remained mostly unknown. Using comparative genomics, we have identified human genes encoding the last four enzymatic steps in CoA biosynthesis: phosphopantothenoylcysteine synthetase (EC ), phosphopantothenoylcysteine decarboxylase (EC ), phosphopantetheine adenylyltransferase (EC ), and dephospho-CoA kinase (EC ). Biological functions of these human genes were verified using a complementation system in Escherichia coli based on transposon mutagenesis. The individual human enzymes were overexpressed in E. coli and purified, and the corresponding activities were experimentally verified. In addition, the entire pathway from phosphopantothenate to CoA was successfully reconstituted in vitro using a mixture of purified recombinant enzymes. Human recombinant bifunctional phosphopantetheine adenylyltransferase/dephospho-CoA kinase was kinetically characterized. This enzyme was previously suggested as a point of CoA biosynthesis regulation, and we have observed significant differences in mRNA levels of the corresponding human gene in normal and tumor cells by Northern blot analysis.