Modification of the NADH of the isoniazid target (InhA) from Mycobacterium tuberculosis

Science

Volumn 279, Issue 5347, 1998, Pages 98-102

  Rozwarski, Denise A ^a   Grant, Gregory A ^a   Barton, Derek H R ^a   Jacobs Jr , William R ^a   Sacchettini, James C ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

ISONIAZID; MYCOLIC ACID; REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE; TUBERCULOSTATIC AGENT;

ARTICLE; BACTERIAL INFECTION; BACTERIUM ISOLATION; DRUG MECHANISM; ENZYME ACTIVE SITE; MASS SPECTROMETRY; MYCOBACTERIOSIS; MYCOBACTERIUM TUBERCULOSIS; NONHUMAN; PRIORITY JOURNAL; TUBERCULOSIS; X RAY CRYSTALLOGRAPHY;

ANTITUBERCULAR AGENTS; BACTERIAL PROTEINS; BINDING SITES; BIOTRANSFORMATION; CRYSTALLOGRAPHY, X-RAY; DRUG RESISTANCE, MICROBIAL; ENOYL-(ACYL-CARRIER-PROTEIN) REDUCTASE (NADH); FATTY ACID SYNTHETASE COMPLEX; ISONIAZID; MASS SPECTROMETRY; MODELS, MOLECULAR; MUTATION; MYCOBACTERIUM TUBERCULOSIS; MYCOLIC ACIDS; NAD; OXIDOREDUCTASES;

ACTINOBACTERIA (CLASS); BACTERIA (MICROORGANISMS); MYCOBACTERIUM; MYCOBACTERIUM TUBERCULOSIS; UNCULTURED ACTINOMYCETE;

EID: 0032472224     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5347.98     Document Type: Article

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43. + at 340 nm in the presence of a fatty acyl-CoA substrate (6). InhA activity decreased by 90% within 2 days and coincided with the reaction mixture turning bright yellow. Crystals of isoniazid-inhibited InhA were produced by combining the incubation mixture in a 1 : 1 ratio with 12% methyl pentane diol, 4% dimethyl sulfoxide, 100 mM Hepes at pH 7.5, and 50 mM sodium citrate at pH 6.5, and placing it in a hanging drop enclosure. Within a week, crystals reached a size of 0.4 × 0.3 × 0.3 mm and were of the same morphology as those used previously to determine the native crystal structure (78).
44. A single crystal was used to collect a data set at room temperature with a MacScience DIP2030 image plate system with double-focusing mirrors coupled to a Rigaku x-ray generator, using a copper rotating anode with a 0.005-mm nickel filter and a 0.5-mm x-ray beam collimator. The Denzo and Scalepack package (26) was used to autoindex, integrate, and scale frames of data. Data collection statistics are listed in Table 1.
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46. 2+ ions) by using a Pharmacia Superdex-75 HR-10/30 gel-filtration column equilibrated in 100 mM triethyl-ammonium acetate, pH 7.0, at a flow rate of 0.5 ml/min. The fractions containing the isoniazid-inhibited InhA were pooled and concentrated to about 2 mg/ml. An enzymatic activity assay confirmed that the purified protein was inactive, which implies that the isonicotinic acyl-NADH was still bound to InhA. Portions of this enzyme preparation (10 to 20 μl) were brought to 100 μl with a solution of 50% methanol and 1% acetic acid in water. These dilutions were infused into a Finnigan LCQ electrospray mass spectrometer at 5 μl/min. Spectra were analyzed in positive mode and averaged for 1 min. The mass value of 770 daltons obtained for the isonicotinic acyl-NADH, present within the isoniazid-inhibited InhA sample, corresponds to the addition of a mass fragment consisting of NADH with one hydrogen removed (mass = 664 daltons) plus a mass fragment consisting of an isonicotinic acyl group derived from isoniazid (mass = 106 daltons).
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61. Financial support was provided by the Welch Foundation and NIH grants GM-45859 and AI-36849. We thank C. Vilcheze and R. Bittman for supplying 2-trans-octenoyl-coenzymeA for InhA activity assays, M. W. Crankshaw for mass spectrometry analysis, members of the Center for Structural Biology at Texas A&M University (http://reddrum.tamu.edu/ vivek/Public) for helpful discussions, and M. Edwards for manuscript preparation assistance. The coordinates have been deposited in the Protein Data Bank with entry number 1zid.