Substrate Specificity and Identification of Functional Groups of Homoserine Kinase from <i>Escherichia coli</i>

Huo, Xuewen; Viola, Ronald E.

doi:10.1021/bi962203z

Cited by 28 publications

(19 citation statements)

References 19 publications

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“…The simplest explanation for these levels of conversion is that transamination of 2 proceeds rapidly but that AAT has a high K M for KHB (so that the reaction effectively terminates at a low concentration of KHB, 20 mM). In contrast, because homoserine 3 is the canonical substrate for HSK it has a much higher affinity, K M <1 mM [55], but the kinetics of the HSK reaction are slower than for the AAT catalyzed step (hence the observation of the homoserine intermediate).…”

Section: Resultsmentioning

confidence: 97%

An Economical Method for Production of 2H,13CH3-Threonine for Solution NMR Studies of Large Protein Complexes: Application to the 670 kDa Proteasome

2012

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NMR studies of very high molecular weight protein complexes have been greatly facilitated through the development of labeling strategies whereby 13CH3 methyl groups are introduced into highly deuterated proteins. Robust and cost-effective labeling methods are well established for all methyl containing amino acids with the exception of Thr. Here we describe an inexpensive biosynthetic strategy for the production of L-[α-2H; β−2H;γ-13C]-Thr that can then be directly added during protein expression to produce highly deuterated proteins with Thr methyl group probes of structure and dynamics. These reporters are particularly valuable, because unlike other methyl containing amino acids, Thr residues are localized predominantly to the surfaces of proteins, have unique hydrogen bonding capabilities, have a higher propensity to be found at protein nucleic acid interfaces and can play important roles in signaling pathways through phosphorylation. The utility of the labeling methodology is demonstrated with an application to the 670 kDa proteasome core particle, where high quality Thr 13C,1H correlation spectra are obtained that could not be generated from samples prepared with commercially available U-[13C,1H]-Thr.

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Section: Resultsmentioning

confidence: 97%

An Economical Method for Production of 2H,13CH3-Threonine for Solution NMR Studies of Large Protein Complexes: Application to the 670 kDa Proteasome

2012

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“…It is not inhibited by shikimate concentrations up to 10 mM, and its K m,ATP (48 Ϯ 4 M) is comparable to the E. coli SK2 apparent K m,ATP (160 M). Homoserine kinase of E. coli (gene thrB), with a K m,homoserine of 140 M, loses up to 70% of its activity when the substrate concentration is increased from 1 to 10 mM (15). The archaeal homoserine kinase RMJ01903 reveals a similar K m,homoserine (188 Ϯ 37 M) but no substrate inhibition up to 10 mM homoserine.…”

Section: Resultsmentioning

confidence: 99%

Archaeal Shikimate Kinase, a New Member of the GHMP-Kinase Family

et al. 2001

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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 (gi1500322 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 (gi1591748), 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 steadystate kinetic parameters similar to those of the corresponding bacterial kinases: K m,shikimate ‫؍‬ 414 ؎ 33 M, K m,ATP ‫؍‬ 48 ؎ 4 M, and k cat ‫؍‬ 57 ؎ 2 s ؊1 for the predicted shikimate kinase and K m,homoserine ‫؍‬ 188 ؎ 37 M, K m,ATP ‫؍‬ 101 ؎ 7 M, 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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“…Prior studies of GHMP family members indicate critical catalytic and substrate binding roles for carboxylate-and hydroxyl-containing side chains (37,(53)(54)(55). Here we changed 10 highly conserved amino acids of PduX that have such side chains and tested the effect of these mutations on structure and kinetic parameters.…”

Section: Discussionmentioning

confidence: 99%

Kinetic and Functional Analysis of l-Threonine Kinase, the PduX Enzyme of Salmonella enterica

Fan

Fromm

Bobik

2009

Journal of Biological Chemistry

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The PduX enzyme of Salmonella enterica is an L-threonine kinase used for the de novo synthesis of coenzyme B 12 and the assimilation of cobyric acid. PduX with an N-terminal histidine tag (His 8 -PduX) was produced in Escherichia coli and purified. The recombinant enzyme was soluble and active. Kinetic analysis indicated a steady-state Ordered Bi Bi complex mechanism in which ATP is the first substrate to bind. Based on a multiple sequence alignment of PduX homologues and other GHMP (galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase) family members, 14 PduX variants having changes at 10 conserved serine/threonine and aspartate/ glutamate sites were constructed by site-directed mutagenesis. Each variant was produced in E. coli and purified. Comparison of the circular dichroism spectra and kinetic properties of the PduX variants with those of the wild-type enzyme indicated that Glu-24 and Asp-135 are needed for proper folding, Ser-99 and Glu-132 are used for ATP binding, and Ser-253 and Ser-255 are critical to L-threonine binding whereas Ser-100 is essential to catalysis, but its precise role is uncertain. The studies reported here are the first to investigate the kinetic and catalytic mechanisms of L-threonine kinase from any organism.The B 12 coenzymes (adenosylcobalamin (AdoCbl) 2 and methylcobalamin) are the largest cofactors known in biology. They are essential for human health and have important metabolic roles in many microbes (1, 2). The B 12 coenzymes are synthesized de novo only by certain prokaryotes and from corrinoid precursors by a broader range of organisms (1, 2). De novo synthesis by prokaryotes is the ultimate source of B 12 , and this process has been extensively studied because of its importance to diverse biological forms and to the commercial production of B 12 as a dietary supplement (1). Salmonella enterica is an important model organism for studies of B 12 synthesis (3, 4). This organism carries out de novo synthesis under anaerobic conditions and assimilates corrinoids such as cobinamide and cobyric acid (Cby) under both aerobic and anaerobic conditions (3, 5). We recently showed that the PduX enzyme of S. enterica is an L-threonine (L-Thr) kinase, which is required for the de novo synthesis of AdoCbl and methylcobalamin and the assimilation of Cby (6). PduX catalyzes the conversion of L-Thr and ATP to ADP and L-threonine-O-3-phosphate (L-Thr-P), a required building block for the de novo synthesis of B 12 (Fig. 1) (6). By sequence similarity, the PduX enzyme of S. enterica belongs to the galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase (GHMP) family (7), which is a novel family of kinases found in metabolic pathways of small molecules. Although there have been no three-dimensional structures of PduX or other L-Thr kinases reported to date, the crystal structures of several GHMP kinases have been solved (8 -22). These structures reveal a unique kinase fold and a novel nucleotide-binding mode that are conserved among members ...

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Substrate Specificity and Identification of Functional Groups of Homoserine Kinase from Escherichia coli

Cited by 28 publications

References 19 publications

An Economical Method for Production of 2H,13CH3-Threonine for Solution NMR Studies of Large Protein Complexes: Application to the 670 kDa Proteasome

An Economical Method for Production of 2H,13CH3-Threonine for Solution NMR Studies of Large Protein Complexes: Application to the 670 kDa Proteasome

Archaeal Shikimate Kinase, a New Member of the GHMP-Kinase Family

Kinetic and Functional Analysis of l-Threonine Kinase, the PduX Enzyme of Salmonella enterica

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