Crystal Structure of Mycobacterium tuberculosis MenB, a Key Enzyme in Vitamin K2 Biosynthesis

There are four main subfamilies of clinically used bicyclic ␤-lactam antibiotics comprised of the penicillins, the cephalosporins, the carbapenems, and the oxacephems. Three inhibitors of the serine ␤-lactamases have also found extensive clinical use and although these compounds are themselves bicyclic ␤-lactams, they possess limited antibiotic activity; hence are used in conjunction with a penicillin. With

Section: Discussionmentioning

confidence: 99%

Structural and Mechanistic Studies on Carboxymethylproline Synthase (CarB), a Unique Member of the Crotonase Superfamily Catalyzing the First Step in Carbapenem Biosynthesis

Sleeman

Sorensen

Batchelar

et al. 2005

“…They all display a typical crotonase family fold as an (␣ 3 ) 2 hexamer with the distinct feature that two of its three C-terminal helices cross the trimer-trimer interface and form a flexible part of the active site within the opposing trimer, as shown for MtbMenB. Using the crystal structure of MtbMenB in complex with acetoacetyl-CoA, the active site residues involved in substrate binding and catalysis have been identified and confirmed through site-directed mutagenesis (31). Surprisingly, a catalytically essential aspartate (Asp-185) in MtbMenB, which occupies a position similar to the highly conserved acidic residue responsible for proton abstraction from the ␣-carbon of the coenzyme A thioester substrates in enoyl-CoA hydratases (crotonase) and enoyl-CoA isomerases (34 -40), is only conserved among a subset of the MenB proteins.…”

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confidence: 99%

“…Surprisingly, a catalytically essential aspartate (Asp-185) in MtbMenB, which occupies a position similar to the highly conserved acidic residue responsible for proton abstraction from the ␣-carbon of the coenzyme A thioester substrates in enoyl-CoA hydratases (crotonase) and enoyl-CoA isomerases (34 -40), is only conserved among a subset of the MenB proteins. In multiple sequence alignment, many MenB proteins contain a glycine at this position (31). Partly due to this lack of sequence conservation, the essential Asp-185 in MtbMenB is proposed to promote the ␣-proton abstraction by a substrate carboxylate in initiation of the intramolecular Claisen condensation, together with another catalytically essential acidic residue, .…”

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confidence: 99%

“…Three-dimensional structures have been solved for MenB proteins from M. tuberculosis (MtbMenB) (31,32), S. aureus (SaMenB) (33), Salmonella typhimurium (StMenB, Protein Data Bank (PDB) ID: 3HO2), and Geobacillus kaustophilus HTA 426 (GkMenB, PDB ID: 2IEX) either in an apo form or in complex with a substrate analog or the product. They all display a typical crotonase family fold as an (␣ 3 ) 2 hexamer with the distinct feature that two of its three C-terminal helices cross the trimer-trimer interface and form a flexible part of the active site within the opposing trimer, as shown for MtbMenB.…”

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confidence: 99%

See 1 more Smart Citation

A Bicarbonate Cofactor Modulates 1,4-Dihydroxy-2-naphthoyl-Coenzyme A Synthase in Menaquinone Biosynthesis of Escherichia coli

Jiang

Chen

Guo

et al. 2010

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 enzymebound bicarbonate allows it to be proposed as a catalytic base responsible for abstraction of the ␣-proton of the thioester substrate in the enzymatic reaction, suggesting a unified catalytic mechanism for all DHNA-CoA synthases.Menaquinone is a lipid soluble naphthoquinone that plays important biological roles. In many bacterial microorganisms, menaquinone serves as an electron transporter in the respiratory chain and is essential for their survival (1, 2). In humans and animals, menaquinone is a vitamin (K2) and is used as an enzyme cofactor involved in post-translational glutamate ␥-carboxylation of proteins in blood coagulation (3, 4), bone metabolism (5), and calcification of arteries and other soft tissues (6). This naphthoquinone is synthesized from the chorismate of the shikimate pathway in microorganisms (1, 2, 7, 8), whereas it has to be acquired from diet or intestinal microflora in humans or animals (9, 10). Due to its absence in humans and animals, menaquinone biosynthesis has been an attractive target for development of antibiotics against a number of important microbial pathogens, such as Mycobacterium tuberculosis and Staphylococcus aureus (11,12).Menaquinone biosynthesis has been most extensively studied in the facultative anaerobe Escherichia coli that uses the naphthoquinone (MK-8) and its demethylated congener (demethylmenaquinone, DMK-8) 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 (15-17), indicating that menaquinone biosynthesis is subject to aerati...

“…9784 (7), we were surprised to find that its closest amino acid sequence homologs in the SwissProt data base were enzymes of the crotonase superfamily. Crotonases are a mechanistically diverse group of enzymes that catalyze a variety of chemical reactions and for which a number of x-ray crystal structures have been reported, including enzymes catalyzing asymmetric double bond hydration (8), decarboxylation (9), dehalogenation (10), the isomerization of double bonds in fatty acids (11), and ring closure to form an aromatic ring (12). Each crotonase superfamily member shares common characteristics despite apparent differences in reaction chemistry.…”

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confidence: 99%

Structure of 6-Oxo Camphor Hydrolase H122A Mutant Bound to Its Natural Product, (2S,4S)-α-Campholinic Acid

Leonard¹,

Grogan

2004

The crotonase homolog, 6-oxo camphor hydrolase (OCH), catalyzes the desymmetrization of bicyclic ␤-diketones to optically active keto acids via an enzymatic retro-Claisen reaction, resulting in the cleavage of a carbon-carbon bond. We have previously reported the structure of OCH (Whittingham, J. L., Turkenburg, J. P., Verma, C. S., Walsh, M. A., and Grogan, G. (2003) J. Biol. Chem. 278, 1744 -1750), which suggested the involvement of five residues, His-45, His-122, His-145, Asp-154, and Glu-244, in catalysis. Here we report mutation studies on OCH that reveal that H145A and D154N mutants of OCH have greatly reduced values of k cat /K m derived from a very large increase in K m for the native substrate, 6-oxo camphor. In addition, H122A has a greatly reduced value of k cat , and its K m is five times that of the wildtype. The location of the active site is confirmed by the 1.9-Å structure of the H122A mutant of OCH complexed with the minor diastereoisomer of (2S,4S)-␣-campholinic acid, the natural product of the enzyme. This shows the pendant acetate of the product hydrogen bonded to a His-145/Asp-154 dyad and the endocyclic carbonyl of the cyclopentane ring hydrogen bonded to Trp-40. The results are suggestive of a base-catalyzed mechanism of C-C bond cleavage and provide clues to the origin of prochiral selectivity by the enzyme and to the recruitment of the crotonase fold for alternate modes of transition state stabilization to those described for other crotonase superfamily members.Enzymatic desymmetrization processes are of great interest to the synthetic organic chemist as they provide quantitative routes to optically active synthetic intermediates from prochiral starting materials (1). We have described the application of the enzyme 6-oxo camphor hydrolase (OCH) 1 to the desymmetrization of bicyclic ␤-diketones for the preparation of optically active keto acids (2), which may be of use in the preparation of chiral synthons of a range of bioactive compounds. This unusual enzymatic process is the biological equivalent of a retro-Claisen or retro-Dieckmann reaction. The carbon-carbon bond of a ␤-dicarbonyl species is cleaved to yield a carboxylic acid and a methylene group. The natural substrate for the enzyme is bornane-2, 6-dione, or 6-oxo camphor 1 (Fig. 1), a natural catabolite of the monoterpene camphor when processed by the natural host strain, Rhodococcus sp. NCIMB 9784 (3). We have previously demonstrated (2) that the enzyme yields a 6:1 diastereomeric ratio of the major product, (2R,4S)-campholinic acid 2 and the diastereoisomer 3 of the (2S,4S) configuration. The cleavage of ␤-dicarbonyl species in nature is mechanistically diverse (4), incorporating, among others, enzymes such as polyvinyl ketone hydrolase (5) from Pseudomonas sp., a serine triad-type hydrolase, and acetylacetone-cleaving enzyme Dke1 (6), a dioxygenase from Acinetobacter johnsonii. On cloning and sequencing the gene encoding OCH, camK, from Rhodococcus sp. 9784 (7), we were surprised to find that its closest amino acid sequence h...