Role of oxygen in the vitamin K-dependent carboxylation reaction: incorporation of a second atom of oxygen 18 from molecular oxygen-18O2 into vitamin K oxide during carboxylase activity

Dowd, Paul; Ham, Seung Wook; Hershline, Roger

doi:10.1021/ja00046a001

Cited by 39 publications

(22 citation statements)

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“…Likewise, in the mass spectrum of the vitamin K oxide product in which the vitamin K-dependent enzymatic carboxylation was carried out under an atmosphere of 1802, the m/e 306 peak is replaced by a peak at mle 308 and the m/e 423 peak is unchanged (26). Therefore, the 180 does indeed occupy the epoxide position in vitamin K oxide isolated from the experiment with the carboxylase under 1802.…”

Section: -mentioning

confidence: 96%

“…Alternatively, oxygenation of the monoanion leads by a similar path to the release of OH-, which, unsolvated in the appropriate hydrophobic enzymatic environment, can also be expected to be a very strong base (Scheme 7). The Labeling studies with 18Q have been highly effective in probing the mechanism (25,26). Model experiments also provide a most useful vehicle for examining these ideas (25,27).…”

Section: Hypothesismentioning

confidence: 99%

“…The 1802 labeling experiment proves to be of highly significant mechanistic import (25,26). If one examines the parent ion cluster at m/e values of 466, 467, and 468 in the mass spectrum of unlabeled vitamin K oxide, the ratio of peak intensities is 100:34:6.…”

Section: -mentioning

confidence: 99%

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Vitamin K and Energy Transduction: A Base Strength Amplification Mechanism

Dowd

Hershline

Ham

et al. 1995

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Energy transfer provides an arrow in the metabolism of living systems. Direct energetic coupling of chemical transformations, such that the free energy generated in one reaction is channeled to another, is the essence of energy transfer, whereas the purpose is the production of high-energy chemical intermediates. Vitamin K provides a particularly instructive example of energy transfer. A key principle at work in the vitamin K system can be termed "base strength amplification." In the base strength amplification sequence, the free energy of oxygenation of vitamin K hydroquinone (vitamin KH2) is used to transform a weak base to a strong base in order to effect proton removal from selected glutamate (Glu) residues in the blood-clotting proteins.

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Section: -mentioning

confidence: 96%

Section: Hypothesismentioning

confidence: 99%

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Vitamin K and Energy Transduction: A Base Strength Amplification Mechanism

Dowd

Hershline

Ham

et al. 1995

Science

Self Cite

134

106

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“…It is known, for instance, that the OtcC anhydrotetracycline oxygenase from S. rimosus requires a specific 5-deazaflavin cofactor (9) which is synthesized in S. lividans but not in E. coli (14). Hypothetical mechanism for the oxidation of TCM A2 to TCM C, modeled on the proposed mechanism of a vitamin K-dependent enzyme (20).…”

mentioning

confidence: 99%

Nucleotide sequences and heterologous expression of tcmG and tcmP, biosynthetic genes for tetracenomycin C in Streptomyces glaucescens

1993

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The nucleotide sequence of the tcmIII, tcmIc, and tcmVII region of the tetracenomycin (TCM) C gene cluster of Streptomyces glaucescens ETH 22794 (GLA.0) revealed the presence of two genes, tcmP and tcmG. The deduced product of tcmG resembles flavoprotein hydroxylases found in several other bacteria, whereas the predicted amino acid sequence oftcmP is not significantly similar to those of any known proteins in the available data-bases. Southern blot hybridization revealed an approximately 180-bp deletion in a tcmIII (tcmG) mutant and a 1,800-bp insertion in a tcmVII (tcmP) mutant. Heterologous expression oftcmG and tcmP in Streptomyces lividans and tcmP in Escherichia coli established that tcmP encodes an O-methyltransferase, catalyzing the methylation of the C-9 carboxy group of TCM E to yield TCM A2, and that tcmG is responsible for the hydroxylation of TCM A2 at positions C-4, C-4a, and C-12a to give TCM C. These are the final two steps of TCM C biosynthesis.Polyketides represent a large family of natural products produced by bacteria, fungi, and plants (29). Considerable interest has been directed into analyzing the biosynthesis of these compounds because of their importance as antibiotics, chemotherapeutics, flavoring agents, and pigments. Understanding the biochemical basis of antibiotic production, in particular, is crucial to establishing rational means for engineering hybrid antibiotic producers (32) or performing tasks like targeted gene disruption that yield a specific and desired product (62). It has been demonstrated that aromatic polyketides like tetracenomycin (TCM) C are synthesized by a type II polyketide synthase (33) that consists of a multiprotein complex (6,22,56). The polyketide backbone of such molecules is subsequently modified by a number of different enzymes, resulting in a vast array of structurally diverse metabolites (29). As a part of the study of the genetics and biochemistry of the formation of the anthracycline antibiotic TCM C (63), we analyzed a region of the tcm cluster (Fig. 1A) characterized by three phenotypically different types of mutants (42,43,45). On the basis of the structure of the compounds accumulated by the Streptomyces glaucescens GLA.0 type III and type VII mutants, it was concluded that this region codes for enzymes that catalyze the last two steps of the TCM C pathway (45). Type III mutants appeared to be blocked in the hydroxylation of TCM A2, whereas type VII mutants were unable to methylate the C-9 carboxy group of TCM E (Fig. 1B) (43), and pTrc99c (1) plasmids are described elsewhere. The ermE* promoter (24) was isolated from pIJ4070 (5). pUC19 was purchased from New England Biolabs (Beverly, Mass.). The Escherichia coli strains used were JM105 and DH5ac (54). The construction of the plasmids used in this study is described in Table 1.Culture conditions and chemicals and other materials. R2YENG liquid medium (pH 6) and agar plates (45, 57) were used for production of TCM C and elloramycin as well as its biosynthetic intermediates. S. glaucescens spores wer...

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“…Vitamin K-dependent g-carboxylation of specific glutamate residues is an important posttranslational modification in proteins, and the carboxylation of glutamate residues is coupled to epoxidation of vitamin K [88]. It has been established that vitamin K-dependent glutamate carboxylase has dioxygenase rather than monooxygenase activity [89,90]. Dioxygenation of reduced vitamin K is thought to proceed through a dioxetane intermediate, cleavage of the O-O bond furnishing the epoxide and an alkoxide anion.…”

Section: Dioxygenasesmentioning

confidence: 99%