Abstract
1,4-Dihydroxy-2-naphthoyl coenzyme A (DHNA-CoA) synthase is a typical crotonase-fold protein catalyzing an intramolecular Claisen condensation in the menaquinone biosynthetic pathway. We have characterized this enzyme from Escherichia coli and found that it is activated by bicarbonate in a concentration-dependent manner. The bicarbonate binding site has been identified in the crystal structure of a virtually identical ortholog (96.8% sequence identity) from Salmonella typhimurium through comparison with a bicarbonate-insensitive orthologue. Kinetic properties of the enzyme and its site-directed mutants of the bicarbonate binding site indicate that the exogenous bicarbonate anion is essential to the enzyme activity. With this essential catalytic role, the simple bicarbonate anion is an enzyme cofactor, which is usually a small organic molecule derived from vitamins, a metal ion, or a metal-containing polyatomic anionic complex. This finding leads to classification of the DHNA-CoA synthases into two evolutionarily conserved subfamilies: type I enzymes that are bicarbonate-dependent and contain a conserved glycine at the bicarbonate binding site; and type II enzymes that are bicarbonate-independent and contain a conserved aspartate at the position similar to the enzyme-bound bicarbonate. In addition, the unique location of the enzyme-bound bicarbonate allows it to be proposed as a catalytic base responsible for abstraction of the α-proton of the thioester substrate in the enzymatic reaction, suggesting a unified catalytic mechanism for all DHNA-CoA synthases.
Highlights
Menaquinone biosynthesis has been most extensively studied in the facultative anaerobe Escherichia coli that uses the naphthoquinone (MK-8) and its demethylated congener as an obligatory electron transporter under anaerobic conditions when the electron acceptor is fumarate, dimethyl sulfoxide, or trimethylamine N-oxide [13, 14]
The naphthoquinone level is significantly up-regulated during the aerobiosis-to-anaerobiosis transition [15,16,17], indicating that menaquinone biosynthesis is subject to aeration control
In accordance with these findings, MenB proteins are divided into two evolutionarily conserved groups with distinct bicarbonate dependence. These findings have demonstrated that the simple bicarbonate anion is able to serve as an enzyme cofactor but have provided new insights into the catalytic mechanism of the menaquinone biosynthetic enzyme. These results identify EcMenB as the only enzyme subjected to external control in the E. coli biosynthesis of menaquinone, which may be involved in the proposed regulation of the pathway by post-transcriptional modulation of enzymatic activity
Summary
Materials—Chemical reagents, including NaHCO3, NH4HCO3, KHCO3, DHNA, chorismate, ␣-ketoglutarate, thiamine diphosphate, coenzyme A, adenosine 5Ј-triphosphate, isopropyl -D-thiogalactopyranoside (IPTG), buffers, and other salts, were purchased from Sigma. The menB genes were amplified from the genomic DNA isolated from E. coli XL1 Blue (Stratagene), Bacillus subtilis 1A 698 (Bacillus Genetic Stock Center), and Mycobacterium smegmatis (ATCC 23037D) and were subcloned into pET32a (Novagen) for overexpression as untagged proteins. The menB gene from a clinical strain of M. tuberculosis was amplified from the genomic DNA sample provided by Professor Pak-Leung Ho of the Hong Kong University and subcloned into a pET28a vector (Novagen) for overexpression as a hexahistidine-tagged protein. A buffered solution containing 50 mM sodium phosphate (pH 7.0), 1–30 M SHCHC, 200 M ATP, 200 M CoA-SH, and 10 mM MgCl2 was added to MenC to a final concentration of 1 M and incubated 15 min at room temperature (23 °C) for complete conversion of SHCHC to OSB (verified by HPLC and UVvisible spectroscopy). The phylogenetic trees were constructed by the Neighbor program in PHYLIP 3.67 under the JonesTaylor-Thornton evolutionary model and were edited using the TreeExplorer software
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