Characterization of the Mycobacterium tuberculosis 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase

TY - JOUR

T1 - Characterization of the Mycobacterium tuberculosis 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase

T2 - potential for drug development

A1 - Eoh,Hyungjin

A1 - Brown,Amanda C.

A1 - Buetow,Lori

A1 - Hunter,William N.

A1 - Parish,Tanya

A1 - Kaur,Devinder

A1 - Brennan,Patrick J.

A1 - Crick,Dean C.

AU - Eoh,Hyungjin

AU - Brown,Amanda C.

AU - Buetow,Lori

AU - Hunter,William N.

AU - Parish,Tanya

AU - Kaur,Devinder

AU - Brennan,Patrick J.

AU - Crick,Dean C.

PY - 2007/12

Y1 - 2007/12

N2 - <p>Mycobacterium tuberculosis utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate and its isomer, dimethylallyl diphosphate, precursors of all isoprenoid compounds. This pathway is of interest as a source of new drug targets, as it is absent from humans and disruption of the responsible genes has shown a lethal phenotype for Escherichia coli. In the MEP pathway, 4-diphosphocytidyl-2-C-methyl-D-erythritol is formed from 2-C-methyl-D-erythritol 4-phosphate (MEP) and CTP in a reaction catalyzed by a 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD). In the present work, we demonstrate that Rv3582c is essential for M. tuberculosis: Rv3582c has been cloned and expressed, and the encoded protein has been purified. The purified M. tuberculosis IspD protein was capable of catalyzing the formation of 4-diphosphocytidyl-2-C-methyl-D-erythritol in the presence of MEP and CTP. The enzyme was active over a broad pH range (pH 6.0 to 9.0), with peak activity at pH 8.0. The activity was absolutely dependent upon divalent cations, with 20 mM Mg2+ being optimal, and replacement of CTP with other nucleotide 5'-triphosphates did not support activity. Under the conditions tested, M. tuberculosis IspD had K-m values of 58.5 mu M for MEP and 53.2 mu M for CTP. Calculated k(cat) and k(cat)/K-m values were 0.72 min(-1) and 12.3 mM(-1) min(-1) for MEP and 1.0 min(-1) and 18.8 mM(-1) min(-1) for CTP, respectively.</p>

AB - <p>Mycobacterium tuberculosis utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate and its isomer, dimethylallyl diphosphate, precursors of all isoprenoid compounds. This pathway is of interest as a source of new drug targets, as it is absent from humans and disruption of the responsible genes has shown a lethal phenotype for Escherichia coli. In the MEP pathway, 4-diphosphocytidyl-2-C-methyl-D-erythritol is formed from 2-C-methyl-D-erythritol 4-phosphate (MEP) and CTP in a reaction catalyzed by a 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD). In the present work, we demonstrate that Rv3582c is essential for M. tuberculosis: Rv3582c has been cloned and expressed, and the encoded protein has been purified. The purified M. tuberculosis IspD protein was capable of catalyzing the formation of 4-diphosphocytidyl-2-C-methyl-D-erythritol in the presence of MEP and CTP. The enzyme was active over a broad pH range (pH 6.0 to 9.0), with peak activity at pH 8.0. The activity was absolutely dependent upon divalent cations, with 20 mM Mg2+ being optimal, and replacement of CTP with other nucleotide 5'-triphosphates did not support activity. Under the conditions tested, M. tuberculosis IspD had K-m values of 58.5 mu M for MEP and 53.2 mu M for CTP. Calculated k(cat) and k(cat)/K-m values were 0.72 min(-1) and 12.3 mM(-1) min(-1) for MEP and 1.0 min(-1) and 18.8 mM(-1) min(-1) for CTP, respectively.</p>

KW - ESCHERICHIA-COLI

KW - ISOPRENOID BIOSYNTHESIS

KW - LETHAL MUTATIONS

KW - CELL-WALL

KW - GENE

KW - 1-DEOXY-D-XYLULOSE

KW - IDENTIFICATION

KW - 5-PHOSPHATE

KW - INTERMEDIATE

KW - MUTAGENESIS

U2 - 10.1128/JB.00925-07

DO - 10.1128/JB.00925-07

M1 - Article

JO - Journal of Bacteriology

JF - Journal of Bacteriology

SN - 0021-9193

IS - 24

VL - 189

SP - 8922

EP - 8927

ER -