Structural characterization of recombinant catalase-peroxidase from Mycobacterium tuberculosis. JUDIT M. NAGY1, DMlTRl SVERGUNZ, MICHEL H.J. KOCHZ, ...
Biochemical Society Transactions (1997) 25 S617
82 Structural characterization of recombinant catalase-peroxidasefrom Mycobacterium tuberculosis
Figure 1. Solutlon X-ray scattering from catalase-peroridase
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JUDIT M. NAGY1, DMlTRl SVERGUNZ, MICHEL H.J. KOCHZ, ANTHONY E.G. CASS1 AND KATHERINE A. BROWN1
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It has long been observed that resistance to isoniazid (INH), the core compound used to treat tuberculosis, is often correlated with reduced levels of catalase activity [1,2]. It has also been confirmed that the presence of active catalaseperoxidase (CP), encoded by the katG gene, is necessary for INH sensitivity in M. tuberculosis [3]. Point mutations or deletions in katG can give rise to clinical isolates with increased levels of INH resistance [4]. The requirement for a katG gene product has been further supported by the observation that transformation of a plasmid harbouring this gene into INH-resistant M. smegmatis strains can restore INH sensitivity [5]. Although it is reasonable to assume that the presence of a functional catalase-peroxidase is required for INH sensitivity in M. tuberculosis, the mechanism of action of INH in the bacterium is not well defined. It has been shown that INH is capable of being oxidized by the katG gene product [6] however, the reactive intermediates responsible for the cellular effects of INH have yet to be identified. CP belongs to a family of bacterial enzymes which are believed to have evolved by a gene duplication [7] involving two modules to produce an 80 kDa subunit. Other catalaseperoxidase enzymes have been successfully purified from a variety of sources. They are generally multimers composed of identical subunits approximately 80 kDa in size. The CP enzymes from Escherichia coli [8] and from M. smegmatis 191 are tetrameric. CP can also be found in dimeric and monomeric form as well. The amino-terminal half of the CP enzyme family shows sequence homology with yeast cytochrome c peroxidase [lo]. The carboxy-terminal half may contain the catalase active site but lacks good sequence homology with known catalase structures. In order to develop a better understanding of the structural properties of CP, small-angle X-ray scattering (SAXS) experiments have been undertaken. Recombinant kutG-encoded M. tuberculosis CP (mtCP) was prepared by overproducing the enzyme in E. coli. The enzyme has been purified to homogeneity according to an established protocol [ l l , 121. mtCP was prepared in a range of concentrations in phosphate buffered saline (PBS). SAXS experiments have been carried out on beamline X33 of the EMBL storage ring DORIS of DESY (Deuthsches Elektronen Synchrotron, Hamburg, Germany). The results are shown in Figure 1 and 2. Over a protein concentration range between 2 and 35 mg/ml, CP appears to assemble as a dimer with an estimated molecular mass of 160 kDa. This result is consistent with gel filtration and native polyacrylamide gel electrophoresis studies which also demonstrated the presence of soluble, active protein in a dimeric state. Analysis of the experimental scattering curve (Figure 1) indicates a radius of gyration of 3.8 nm and an excluded volume of 306 nm3. This data has also been used to construct a molecular envelope (Figure 2) using a shape determination algorithm which utilises the two-fold symmetry of this molecule [131. This envelope suggests a "head-to-head" assembly of mtCP and is being used to guide the
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Figure 2. Model of catalase-peroxidase dimer conaisting of two monomers.
construction of a homology-based model of this protein. Acknowledgements: This work has been supported by the BBSRC and the Commission of European Communities. 1. Middlebrook, G. (1954) Am. Rev. Tuberc. 69,471-472 2. Youatt, J. (1969) h e r . Rev. Resp. Dis., 99,729-749 3. Zhang, Y., Heym, B., Allen, B., Young, D. & Cole, S. (1992) Nature 358,591-593 4. Heym, B., Alzari, P. M., Honore, N., & Cole, S. T. (1995) Mol Microbioll5,235-245 5. Zhang, Y., Garbe, T., & Young, D. (1993) Mol Microbiol8, 521-524 6. Johnsson, K. & Shultz, P.G. (1994) J. Am. Chem. SOC.116, 7425-7526 7. Welinder, K.G. (1991) Biochim. Biophys. Acta 1080,215220 8. Triggs-Raine, B.L., Doble, B.W., Mulvey, M.R., Sorby, P.A. and Loewen, P.C. (1988)J. Bactenol. 170,4415-4419 9. Marcinkeviciene, J.A., Magliozzo, R.S. & Blanchard, J.S. (1995) J. Biol. Chem. 270,22290-22295 10. Finzei,-B.C., Poulos, T.L. & Kraut, J. (1984) J. Biol. Chem. 259,13027-13036 11.Nagy, J.M., Cass, A.E.G. & Brown, K.A. (1995) Biochem. Soc. Trans. 23,152s 12. Nagy, J.M., Cass, A.E.G. & Brown, K.A. (manuscript submitted ) 13. Svergun, D.I., Volkov, V.V., Kozin, M.B. & Stuhrmann, H.B. (1996) Acta Cryst. A52,419-426
