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. 2001 Jan;183(1):292-300.
doi: 10.1128/JB.183.1.292-300.2001.

Archaeal shikimate kinase, a new member of the GHMP-kinase family

M Daugherty  1 , V Vonstein, R Overbeek, A Osterman
Affiliations

Archaeal shikimate kinase, a new member of the GHMP-kinase family

M Daugherty et al. J Bacteriol. 2001 Jan.

Abstract

Shikimate kinase (EC 2.7.1.71) is a committed enzyme in the seven-step biosynthesis of chorismate, a major precursor of aromatic amino acids and many other aromatic compounds. Genes for all enzymes of the chorismate pathway except shikimate kinase are found in archaeal genomes by sequence homology to their bacterial counterparts. In this study, a conserved archaeal gene (gi1500322 in Methanococcus jannaschii) was identified as the best candidate for the missing shikimate kinase gene by the analysis of chromosomal clustering of chorismate biosynthetic genes. The encoded hypothetical protein, with no sequence similarity to bacterial and eukaryotic shikimate kinases, is distantly related to homoserine kinases (EC 2.7.1.39) of the GHMP-kinase superfamily. The latter functionality in M. jannaschii is assigned to another gene (gi591748), in agreement with sequence similarity and chromosomal clustering analysis. Both archaeal proteins, overexpressed in Escherichia coli and purified to homogeneity, displayed activity of the predicted type, with steady-state kinetic parameters similar to those of the corresponding bacterial kinases: K(m,shikimate) = 414 +/- 33 microM, K(m,ATP) = 48 +/- 4 microM, and k(cat) = 57 +/- 2 s(-1) for the predicted shikimate kinase and K(m,homoserine) = 188 +/- 37 microM, K(m,ATP) = 101 +/- 7 microM, and k(cat) = 28 +/- 1 s(-1) for the homoserine kinase. No overlapping activity could be detected between shikimate kinase and homoserine kinase, both revealing a >1,000-fold preference for their own specific substrates. The case of archaeal shikimate kinase illustrates the efficacy of techniques based on reconstruction of metabolism from genomic data and analysis of gene clustering on chromosomes in finding missing genes.

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Figures

FIG. 1
FIG. 1
Pathway reconstruction from genomic data, and chromosomal clustering of chorismate biosynthetic genes. Gene names shown in italic are those of E. coli. Orthologous ORFs found in other genomes are shown by RID numbers from the WIT database. Shaded boxes within a genome row represent proximity on the chromosome.
FIG. 2
FIG. 2
Chorismate biosynthesis pathway in E. coli (modified from reference 34). Enzyme names and corresponding E. coli genes are displayed.
FIG. 3
FIG. 3
Alignment of selected chromosomal contigs containing chorismate biosynthetic genes, modified from data produced by the WIT tool Pinned Regions to visualize gene clustering on the chromosome. The display is created by aligning one specific gene from a number of organisms and depicting other orthologous genes that are conserved in the neighborhood at least in two different genomes. Contigs are aligned by 3-dehydroquinate synthase (the second gene of the pathway). ORFs with sequence similarity are outlined using the same pattern, and those with assigned functions in chorismate biosynthesis are marked with a number corresponding to the step in the pathway as in Fig. 1. Patterns are retained within gene fusions to show regions of homology with corresponding genes. The two genes marked with question marks are those predicted to encode an archaeal shikimate kinase.
FIG. 4
FIG. 4
Amino acid sequence alignment of archaeal shikimate kinases. Conserved residues are highlighted. The segments bracketed with numbers 1 and 2 correspond to sites that are similarly conserved in homoserine kinase and involved in forming an ATP-binding site (42).
FIG. 5
FIG. 5
Initial rate plots obtained for the shikimate kinase RMJ07785 versus shikimate concentration (A) and for the homoserine kinase RMJ01903 versus homoserine concentration (B). Symbols represent experimental data at various concentrations of ATP: 14 μM (▵), 35 μM (◊), 70 μM (□), 140 μM (▿), and 350 μM (○). Curves show global fits of the data using equation 2 for the shikimate kinase (parameters of the fit were KA = 48 ± 4 μM, KB = 414 ± 33 μM, and kcat = 57 ± 2 s−1) and equation 1 for the homoserine kinase (parameters of the fit were KA = 101 ± 7 μM, KB = 188 ± 37 μM, K1A = 475 ± 112 μM, and kcat = 28 ± 1 s−1).
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