Structure and Function of Carbonic Anhydrases from Mycobacterium tuberculosis

Abstract

Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate. This activity is universally required for fatty acid biosynthesis as well as for the production of a number of small molecules, pH homeostasis, and other functions. At least three different carbonic anhydrase families are known to exist, of which the α-class found in humans has been studied in most detail. In the present work, we describe the structures of two of the three β-class carbonic anhydrases that have been identified in Mycobacterium tuberculosis, i.e. Rv1284 and Rv3588c. Both structures were solved by molecular replacement and then refined to resolutions of 2.0 and 1.75 Å, respectively. The active site of Rv1284 is small and almost completely shielded from solvent, whereas that of Rv3588c is larger and quite open to solution. Differences in coordination of the active site metal are also observed. In Rv3588c, an aspartic acid side chain displaces a water molecule and coordinates directly to the zinc ion, thereby closing the zinc coordination sphere and breaking the salt link to a nearby arginine that is a feature of Rv1284. The two carbonic anhydrases thus exhibit both of the metal coordination geometries that have previously been observed for structures in this family. Activity studies demonstrate that Rv3588c is a completely functional carbonic anhydrase. The apparent lack of activity of Rv1284 in the present assay system is likely exacerbated by the observed depletion of zinc in the preparation. Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate. This activity is universally required for fatty acid biosynthesis as well as for the production of a number of small molecules, pH homeostasis, and other functions. At least three different carbonic anhydrase families are known to exist, of which the α-class found in humans has been studied in most detail. In the present work, we describe the structures of two of the three β-class carbonic anhydrases that have been identified in Mycobacterium tuberculosis, i.e. Rv1284 and Rv3588c. Both structures were solved by molecular replacement and then refined to resolutions of 2.0 and 1.75 Å, respectively. The active site of Rv1284 is small and almost completely shielded from solvent, whereas that of Rv3588c is larger and quite open to solution. Differences in coordination of the active site metal are also observed. In Rv3588c, an aspartic acid side chain displaces a water molecule and coordinates directly to the zinc ion, thereby closing the zinc coordination sphere and breaking the salt link to a nearby arginine that is a feature of Rv1284. The two carbonic anhydrases thus exhibit both of the metal coordination geometries that have previously been observed for structures in this family. Activity studies demonstrate that Rv3588c is a completely functional carbonic anhydrase. The apparent lack of activity of Rv1284 in the present assay system is likely exacerbated by the observed depletion of zinc in the preparation. The World Health Organization estimates that 1.8 billion people are infected with Mycobacterium tuberculosis, with 8 million new cases and 2 million deaths per year. The so-called “short-term” antibiotic treatment currently available lasts 6 months and does not seem to offer much hope for eradicating the disease in the long run. In the milestone publication of the M. tuberculosis genome sequence, Cole et al. (1Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry III, C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver K. Osborne J. Quail M.A. Rajandream M.-A. Rogers J. Rutter S. Seeger K. Skelton J. Squares R. Squares S. Sulston J.E. Taylor K. Whitehead S. Barrell B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6522) Google Scholar) were able to assign functions to some 60% of the gene products. Subsequent studies have attempted to identify genes that are likely to be required for bacterial survival and infection (e.g. studies by Sassetti and Rubin (2Sassetti C.M. Rubin E.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12989-12994Crossref PubMed Scopus (1050) Google Scholar) and Sassetti et al. (3Sassetti C.M. Boyd D.H. Rubin E.J. Mol. Microbiol. 2003; 48: 77-84Crossref PubMed Scopus (2007) Google Scholar)). We have used such investigations as the basis for identifying M. tuberculosis gene products that are interesting subjects for detailed structural and functional studies. The gene Rv1284 is annotated at the Pasteur Institute TubercuList server (genolist.pasteur.fr/TubercuList) as coding for a conserved hypothetical protein of unknown function, that is, it is found in related species and presumably has an essential role in their survival, although it is not clear what function is involved. Furthermore, Rv1284 was judged to be essential by Himar1-based transposon mutagenesis in strain H37Rv (3Sassetti C.M. Boyd D.H. Rubin E.J. Mol. Microbiol. 2003; 48: 77-84Crossref PubMed Scopus (2007) Google Scholar), and its transcription was highly up-regulated under the starvation conditions used to model persistent bacteria (4Betts J.C. Lukey P.T. Robb L.C. McAdam R.A. Duncan K. Mol. Microbiol. 2002; 43: 717-731Crossref PubMed Scopus (1139) Google Scholar). Phylogenetic studies of putative carbonic anhydrases (CAs) 1The abbreviations used are: CA, carbonic anhydrase; TAPS, 3-[tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid; SSPE, saline/sodium phosphate/EDTA; r.m.s.d., root mean square deviation. (EC 4.2.1.1) suggested that Rv1284 in fact encodes a β-CA and uncovered distant relationships to two other M. tuberculosis proteins, those produced by the Rv3588c gene and the C-terminal region of Rv3273 (5Smith K.S. Jakubzick C. Whittam T.S. Ferry J.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15184-15189Crossref PubMed Scopus (319) Google Scholar). Rv3588c has been identified as being required for mycobacterial growth in vivo by Sassetti and Rubin (2Sassetti C.M. Rubin E.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12989-12994Crossref PubMed Scopus (1050) Google Scholar), whereas Rv3273 is not essential (2Sassetti C.M. Rubin E.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12989-12994Crossref PubMed Scopus (1050) Google Scholar, 3Sassetti C.M. Boyd D.H. Rubin E.J. Mol. Microbiol. 2003; 48: 77-84Crossref PubMed Scopus (2007) Google Scholar). Human carbonic anhydrases belong to the α-class, which has a completely different structure; both α- and β-classes, however, are comprised of zinc-based enzymes (see Ref. 6Lindskog S. Liljas A. Curr. Opin. Struct. Biol. 1993; 3: 915-920Crossref Scopus (58) Google Scholar for a review of carbonic anhydrase structures). This difference as well as the success of drug design for the α-CAs (reviewed in Ref. 7Supuran C.T. Scozzafava A. Casini A. Med. Res. Rev. 2003; 23: 146-189Crossref PubMed Scopus (1159) Google Scholar) prompted us to carry out a detailed analysis of the mycobacterial enzymes. In the present publication we report structural and functional studies of the Rv1284 and Rv3588c gene products. The results demonstrate that Rv3588c is indeed a β-CA and that Rv1284 is a β-CA-like protein. Cloning, Protein Expression, and Purification—The sequences corresponding to the open reading frames of Rv1284 and Rv3588c were amplified by PCR from M. tuberculosis DNA strain H37Rv (1Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry III, C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver K. Osborne J. Quail M.A. Rajandream M.-A. Rogers J. Rutter S. Seeger K. Skelton J. Squares R. Squares S. Sulston J.E. Taylor K. Whitehead S. Barrell B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6522) Google Scholar) with Pfu polymerase using the primer pair 5′-GTGACGGTTACCGACGACTACCTG-3′ (Rv1284.forward) and 5′-TATGGTTATTGCCATCTATGCAAC-3′ (Rv1284.reverse) for Rv1284 and 5′-GTGAGGCATGCCCAACACCAATCCGGTA-3′ (Rv3588c.forward) and 5′-TCCCGGGTACCTATTAGACCTCCTCGCCGATGTT-3′ (Rv3588c.reverse) for Rv3588c. A second PCR to introduce an N-terminal His6 tag into each construct was performed with the same reverse primer and a new forward primer, Rv1284.his (5′-ATGGCCCATCATCATCATCATCATTCTGGTACGGTTACCG-ACGACTACCTG-3′) and Rv3588c.his (5′-ATGGCTCATCATCATCATCATCATGGTCCCAACACCAATCCGGTAGCCGCG-3′), respectively, using the products from the first PCRs as templates. The PCR products were incubated with deoxynucleotide triphosphates and Taq polymerase to create an A-overhang, purified, and ligated into vector pCR®T7 using the TOPO® Cloning pCR® kit (Invitrogen). The resulting plasmids were transformed into Top10 cells (Invitrogen) by heat shock and grown on Luria-Bertani agar plates containing 50 μg/ml ampicillin. Positive clones were transformed into Escherichia coli BL21/AI in which protein was overexpressed after induction with 0.02% arabinose at 37 °C for 2 h. Cells were harvested by centrifugation. Cell pellets were washed with 1× SSPE buffer (150 mm NaCl, 10 mm NaH2PO4, pH 7.5, and 1 mm EDTA) and stored at –70 °C. All clones were verified by DNA sequencing. For purification, thawed cells were re-suspended in native lysis buffer (50 mm NaH2PO4, pH8, 300mm NaCl, 20 mm imidazole, and 10% glycerol) with 0.01 mg/ml RNase, 0.02 mg/ml DNase, and 1 mm phenylmethylsulfonyl fluoride and lysed using a were incubated with for at was then and protein was with mm The protein were to using a buffer containing mm NaCl, 20 mm pH 7.5, and For the buffer also The protein were by Protein were using with a molecular of 10 buffer was 10 mm pH 7.5, and mm for Rv1284 and 20 mm pH 7.5, mm NaCl, and for Rv3588c. and activity was as at using a of previously J. Biol. PubMed Google Scholar). A mm of was by the in and then it with The was by of 50 mm pH 7.5, to 1 of this and the in of was and the was for an A was in each of carbonic anhydrase was used as a Carbonic anhydrase activity was by in pH the with a J. Biol. PubMed Google Scholar). were performed at °C. The used were mm mm 7.5, and both also mm to a of the The was by of of water to of a containing protein and were as for the were at the at using a with with of analysis was performed by for which was by and were grown by the and of Protein and Scholar) at 20 °C. The of 2 of protein with 2 of containing pH 7.5, and after 1 to the was to a with and then in were at at of the in were with 1999; Scholar) and and in of on Scholar) using the Scholar). Rv3588c was using the conditions for were as 1 of protein was with 1 of containing mm and mm at pH to mm in in and be directly from the were at at at the in The were and using and PubMed Scopus Google Scholar). for both are in and Rv1284 was solved by molecular replacement in J. Scopus Google Scholar) using a of the carbonic anhydrase from K.S. Ferry J.G. J. Biol. PubMed Scopus Google Scholar) as a for the All with were whereas other and were to using M. for of F. The Scholar). The first of both in the were from the a in the native the same was used to a second was then performed in E.J. Scopus Google Scholar) with each molecule as a in the M. 2003; Scopus Google Scholar) was used to an not present in the model and with the M. tuberculosis in the model in M. Scopus Google Scholar), and to 2.0 Å, the M. tuberculosis be in were then and the with of and of the was used to a Scopus Google Scholar) for the of and of as in and in of of in of from from Ref. using a as in and in from Ref. R.A. R. Scopus Google using a PubMed Scopus Google Scholar). in a new Rv3588c was solved by molecular replacement using J. M. T. 1998; Scopus Google Scholar). The model was from the two structures in the Protein the Protein Protein Sci. PubMed Scopus Google Scholar) with in a acid and Protein S. T. M. T. T. S. T. J. Biol. PubMed Scopus Google Scholar) with in a acid The model of chain containing in Both and were the was found in analysis that the produced a with a molecule and to was by of and with protein apparent in the of This was as a by the the of the and water was of its in the the coordination and are with this this is distant from the active site and we it functional The for both structures are in coordinates and have been at the Protein J. Res. PubMed Scopus Google Scholar) with and of Rv1284 and Rv1284 and Rv3588c were purified, and and their structures were by molecular Both structures have been refined to with the in The Rv1284 two in the each of which is of a a Rv3588c, on the other a that a by the a The Rv1284 for the sequence, i.e. from 1 to Rv3588c, however, has for and Both structures have the β-CA from of a in which are and The is and to the by a reverse The are of different and in the two The is and in both The a on the of the The is much containing two in Rv1284 and in Rv3588c. of on the in both The of the the of the to with the related and In the first on the of the other as was observed in three of the known β-CA structures K.S. Ferry J.G. J. Biol. PubMed Scopus Google Scholar, Protein Sci. PubMed Scopus Google Scholar, S. T. M. T. T. S. T. J. Biol. PubMed Scopus Google Scholar, J. PubMed Google Scholar) (see of the in Rv3588c we out the that its first in the same the of the that it is not The of Rv1284 and Rv3588c be such that have an of PubMed Scopus Google Scholar), with their of The structural is observed in the as active site of a β-CA a at the C-terminal of its as has been observed for other J. to Protein Scholar). of the present metal both structures at the a zinc in an active β-CA In each we have this as a zinc although the in Rv3588c is that in Rv1284 and respectively, with both structures of of the active site is, however, different in the two In of the metal are by of the although much of the active site is by the other in in the other chain in the the and and in the long the N-terminal to and a water molecule the metal ion, whereas the and form a salt The of the in Rv1284 are thus to those by and for the from J. PubMed Google Scholar). The of the with chain whereas the of with a chain The water a with the of A molecule is also in the active site of although it is not to the metal The Rv1284 active site is small and almost completely shielded from its is a for that of the observed other side from the second In Rv3588c, the active site displaces the water molecule and coordinates directly to the zinc ion, thus breaking a salt link to and The two M. tuberculosis exhibit both of the coordination for K.S. Ferry J.G. J. Biol. PubMed Scopus Google Scholar, Protein Sci. PubMed Scopus Google Scholar, S. T. M. T. T. S. T. J. Biol. PubMed Scopus Google Scholar, J. PubMed Google Scholar). is an in the active site of Rv3588c, which is from the this is conserved in three of the other The Rv3588c active site is much open that of Rv1284 and directly to the This difference be to the lack of an the N-terminal and In Rv3588c the from at the zinc the with from the by The of the protein in Rv1284 the at the metal by the by from the and the is also out of the of the to β-CA structures have previously been in the be to the Rv1284 with of using PubMed Scopus Google Scholar) (see The of the is in the of the M. to which it has to Rv3588c that it is much to the other three structures of Rv1284 with other β-CA number of in the of a in conserved of in root mean square to Protein to to to to tuberculosis, number of in the of a in conserved of in root mean square to Protein in a new of Rv3588c with other β-CA number of in the of a in conserved of in root mean square to Protein number of in the of a in conserved of in root mean square to Protein in a new The β-CA structures are on Rv1284 in The that the and two of the that on it the and are well although is a of in other difference the N-terminal In as well as the S. T. M. T. T. S. T. J. Biol. PubMed Scopus Google Scholar), J. PubMed Google Scholar), and coli Protein Sci. PubMed Scopus Google Scholar) this on the of the other although the coordinates not form a In the from M. K.S. Ferry J.G. J. Biol. PubMed Scopus Google Scholar), the on the of other in the All of the structures as as the conserved aspartic acid in Rv1284 the of the and for the two M. tuberculosis proteins, are in the region and The C-terminal of this long is, however, on the that on in The reverse and conserved in some of structures that are not present in the family. For the in the structures of Protein and are whereas in the other the is In two of the previously structures from and M. three protein side and a water molecule the active site zinc exhibit geometries that are to that observed in Rv1284. The in from at the zinc The other two structures coli and exhibit the coordination observed for Rv3588c. 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Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate. This of a into a is to such enzymes are thus in At least three different families are known to exist, and of which the α-class has been studied in the most S. PubMed Scopus Google Scholar). the fact that the three families are in and are comprised of enzymes. All in the are of the α-class, and the is a drug The for to the active site zinc and is used for the treatment of PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). The α-class have also been found in and enzymes have not been found in the have been observed in a of other In an a a molecule to form a that is by a new water molecule S. PubMed Scopus Google Scholar). to the buffer then the A detailed molecular has been that the of carbon dioxide in a to the in the an role as the and a from the metal to a of water S. PubMed Scopus Google Scholar). The first structural on an different protein and the of its of active of from J. PubMed Google Scholar) and S. T. M. T. T. S. T. J. Biol. PubMed Scopus Google Scholar) different coordination of the metal at the active In the the protein three whereas in the a nearby aspartic acid a protein side chain this was conserved in the in a salt link with a nearby Subsequent of the from M. K.S. Ferry J.G. J. Biol. PubMed Scopus Google Scholar) and coli Protein Sci. PubMed Scopus Google Scholar) of the two in this of is structural that a β-CA coordination A to that for the α-CAs has been suggested for the β-class the of a on the carbon dioxide and from a new water J. PubMed Google Scholar). et al. S. T. M. T. T. S. T. J. Biol. PubMed Scopus Google Scholar) have a on this in which the observed in is directly involved. The that this functions as a that a water molecule and that the resulting displaces the as the zinc in the of the The of this side chain and the arginine of and in both of which are conserved in the have been by mutagenesis for the M. K.S. C. Ferry J.G. J. 2002; PubMed Scopus Google Scholar). The the not to an that a role for the in the A much larger in was observed for the which was to mean that the of the salt coordinates the with this the a activity the of an buffer activity of both the and the that this buffer on the role of the in the mutagenesis of two side in the active site of the to and in Protein suggested that are for in that C. J.E. 2002; PubMed Scopus Google Scholar). are not conserved in the β-CA family. structural studies of two M. tuberculosis gene Rv1284 and Rv3588c, that are both functional this for Rv3588c, although not for Rv1284 analysis that the lack of activity is with a zinc depletion at the active site of Rv1284. This is by the Rv1284 the of the metal is that observed in Rv3588c. zinc the protein to the tag in the present construct as an and thus the observed we are to the construct in the to protein the Both structural L. C. Res. PubMed Scopus Google Scholar) and J. Mol. Biol. 1999; PubMed Scopus Google Scholar) of the structural that the of Rv1284 is with a Rv3588c are grown at a pH at which the has The observed to the that the has side the active site zinc The β-CA structures known us to the structural of this of and to the with a from three to protein side chain coordination at the The structures that the active site metal of the β-CA enzymes is in a is in the family. the of the zinc of the β-CA from the almost site in Rv1284 to the M. which is open to and indeed to a molecule of buffer K.S. Ferry J.G. J. Biol. PubMed Scopus Google Scholar). The is by the the first in the to the first In Rv1284 and the J. PubMed Google Scholar) this the active and a side chain and the The of the has been suggested to with the carbon dioxide as well as the bicarbonate. In the side chain the active site to form a The is with the of at and a water molecule at This water molecule is in by with chain and A from this water it to the of the this is from the zinc we that is a model for the of carbon dioxide to the active site of this β-CA (see Rv3588c the of Rv1284 of and to the of β-CA enzymes with open active the that the in Rv1284 the in in for the A. C. J.E. 2002; PubMed Scopus Google Scholar). dioxide is in fatty acid biosynthesis as well as in the of small et al. C. M. S. A. J. 2003; PubMed Scopus Google Scholar) have that the β-CA by the gene is required for the growth of coli in it is that the for is to be produced by an hydration in a are not have at least α-class C.T. Scozzafava A. Casini A. Med. Res. Rev. 2003; 23: 146-189Crossref PubMed Scopus (1159) Google Scholar), studies of two of the three that have been identified in M. tuberculosis demonstrate that enzymes have a number of and presumably have different functional studies have suggested that Rv1284 has a The of this protein to the from the M. interesting in this although it is not clear which of this are most The second mycobacterial Rv3588c, to be in mycobacterial survival, although its relationships to the and bacterial enzymes it a for drug We that such in the for new that of the and the α-class found in We Cole of the Pasteur for M. tuberculosis