Contrasting evolutionary rates in the duplicate chaperonin genes of Mycobacterium tuberculosis and M. leprae

A phylogenetic analysis of chaperonin (heat shock protein 60) sequences from prokaryotes and eukaryotes indicated that a single gene duplication event in the common ancestor of Mycobacterium tuberculosis, M. leprae, and Streptomyces albus gave rise to the duplicate chaperonin genes found in these species (designated HSP65 and GroEL in the mycobacterial species). Comparison of rates of synonymous and nonsynonymous nucleotide substitution in different gene regions suggested that the 5' end of the HSP65 gene was homogenized by an ancient recombination event between M. tuberculosis and M. leprae. In S. albus, the two duplicated chaperonin genes have evolved at essentially the same rate. In both M. tuberculosis and M. leprae, however, the GroEL gene has evolved considerably more rapidly at nonsynonymous nucleotide sites than has the HSP65 gene. Because this difference is not seen at synonymous sites, it must be due to a difference in selective constraint on the proteins encoded by the two genes, rather than to a difference in mutation rate. The difference between GroEL and HSP65 is striking in regions containing epitopes recognized by T cells of the vertebrate host; in certain cross-reactive epitopes conserved across all organisms, nonsynonymous sites in GroEL have evolved twice as fast as those in HSP65. It is suggested that these differences are correlated with differences in the way in which the duplicate chaperonins of M. tuberculosis and M. leprae interact with the host immune system.      

Organization and nucleotide sequences of the genes encoding the biotin carboxyl carrier protein and biotin carboxylase protein of Pseudomonas aeruginosa acetyl coenzyme A carboxylase.

The genetic organization of the Pseudomonas aeruginosa acetyl coenzyme A carboxylase (ACC) was investigated by cloning and characterizing a P. aeruginosa DNA fragment that complements an Escherichia coli strain with a conditional lethal mutation affecting the biotin carboxyl carrier protein (BCCP) subunit of ACC. DNA sequencing and RNA blot hybridization studies indicated that the P. aeruginosa accB (fabE) homolog, which encodes BCCP, is part of a 2-gene operon that includes accC (fabG), the structural gene for the biotin carboxylase subunit of ACC. P. aeruginosa homologs of the E. coli accA and accD, encoding the alpha and beta subunits of the ACC carboxyltransferase, were identified by hybridization of P. aeruginosa genomic DNA with the E. coli accA and accD. Data are presented which suggest that P. aeruginosa accA and accD homologs are not located either immediately upstream or downstream of the P. aeruginosa accBC operon. In contrast to E. coli, where BCCP is the only biotinylated protein, P. aeruginosa was found to contain at least three biotinylated proteins.     

The genes encoding the biotin carboxyl carrier protein and biotin carboxylase subunits of Bacillus subtilis acetyl coenzyme A carboxylase, the first enzyme of fatty acid synthesis.

The genes encoding two subunits of acetyl coenzyme A carboxylase, biotin carboxyl carrier protein, and biotin carboxylase have been cloned from Bacillus subtilis. DNA sequencing and RNA blot hybridization studies indicated that the B. subtilis accB homolog which encodes biotin carboxyl carrier protein, is part of an operon that includes accC, the gene encoding the biotin carboxylase subunit of acetyl coenzyme A carboxylase.     

Expression and characterization of the carboxyl esterase Rv3487c from Mycobacterium tuberculosis

Rv3487c (lipF), a member of the lipase family of Mycobacterium tuberculosis, is related to virulence of this pathogen. Real-time RT-PCR analysis indicated that Rv3487c was induced at low pH in M. tuberculosis cultured in vitro. The gene of Rv3487c was cloned and expressed as fusion protein in Escherichia coli. After removal of the N-terminal domain of the fusion partner by enterokinase treatment, the effect of pH, temperature, and detergents on the purified enzyme activity and stability was characterized. Rv3487c could efficiently hydrolyze short chain esters. The catalytic triad of Rv3487c consists of residues Ser90, Glu189, and His219 as demonstrated by amino acid sequence alignment, three-dimensional modeling, and site-directed mutagenesis.     

Saccharopolyspora hirsuta 367 encodes clustered genes similar to ketoacyl synthase,ketoacyl reductase,acyl carrier protein,and biotin carboxyl carrier protein

The actI gene, encoding a component of the actinorhodin polyketide synthase of Streptomyces coelicolor, was used to identify and clone a homologous 11.7 kb BamHI DNA fragment from Saccharopolyspora hirsuta 367. The cloned fragment complemented actinorhodin production in a strain of Streptomyces coelicolor bearing a mutant actI gene. The DNA sequence of a 5.1 kb fragment revealed 6 open reading frames (ORF). ORF1 does not resemble any known DNA or deduced protein sequence, while the deduced protein sequence of ORF2 resembles that of biotin carboxyl carrier proteins. Based on the similarity to deduced protein sequences from cloned genes of polyketide producers, ORF3 would code for a ketoreductase, ORF4 and ORF5 for the putative heterodimeric β-ketoacyl synthase, and ORF6 for an acyl carrier protein.     

Expression, purification, and characterization of the Mycobacterium tuberculosis acyl carrier protein, AcpM.

Mycolic acids are generated in Mycobacterium tuberculosis as a result of the interaction of two fatty acid biosynthetic systems: the multifunctional polypeptide, FASI, in which the acyl carrier protein (ACP) domain forms an integral part of the polypeptide, and the dissociated FASII system, which is composed of monofunctional enzymes and a discrete ACP (AcpM). In order to characterize enzymes of the FASII system, large amounts of AcpM are required to generate substrates such as holo-AcpM, malonyl-AcpM and acyl-AcpM. The M. tuberculosis acpM gene was overexpressed in Escherichia coli and AcpM purified, yielding approximately 15-20 mg/l of culture. Analysis of AcpM by mass spectrometry, N-terminal sequencing, amino acid analysis, and gas chromatography indicated the presence of three species, apo-, holo-, and acyl-AcpM, the former comprising up to 65% of the total pool. The apo-AcpM was purified away from the in vivo generated holo- and acyl-forms, which were inseparable and heterogeneous with respect to acyl chain lengths. Once purified, we were able to convert apo-AcpM into holo- and acyl-forms. These procedures provide the means for the preparation of the large quantities of AcpM and derivatives needed for characterization of the purified enzymes of the mycobacterial FASII system.     

Comparison of the UDP-N-acetylmuramate:L-alanine ligase enzymes from Mycobacterium tuberculosis and Mycobacterium leprae

In the peptidoglycan of Mycobacterium leprae, L-alanine of the side chain is replaced by glycine. When expressed in Escherichia coli, MurC (UDP-N-acetyl-muramate:L-alanine ligase) of M. leprae showed K(m) and V(max) for L-alanine and glycine similar to those of Mycobacterium tuberculosis MurC, suggesting that another explanation should be sought for the presence of glycine.     

The biotin domain peptide from the biotin carboxyl carrier protein of Escherichia coli acetyl-CoA carboxylase causes a marked increase in the catalytic efficiency of biotin carboxylase and carboxyltransferase relative to free biotin.

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of long-chain fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase activity, a biotin carboxyl carrier protein, and a carboxyltransferase activity. The C-terminal 87 amino acids of the biotin carboxyl carrier protein (BCCP87) form a domain that can be independently expressed, biotinylated, and purified (Chapman-Smith, A., Turner, D. L., Cronan, J. E., Morris, T. W., and Wallace, J. C. (1994) Biochem. J. 302, 881-887). The ability of the biotinylated form of this 87-residue protein (holoBCCP87) to act as a substrate for biotin carboxylase and carboxyltransferase was assessed and compared with the results with free biotin. In the case of biotin carboxylase holoBCCP87 was an excellent substrate with a K(m) of 0.16 +/- 0.05 mM and V(max) of 1000.8 +/- 182.0 min(-1). The V/K or catalytic efficiency of biotin carboxylase with holoBCCP87 as substrate was 8000-fold greater than with biotin as substrate. Stimulation of the ATP synthesis reaction of biotin carboxylase where carbamyl phosphate reacted with ADP by holoBCCP87 was 5-fold greater than with an equivalent amount of biotin. The interaction of holoBCCP87 with carboxyltransferase was characterized in the reverse direction where malonyl-CoA reacted with holoBCCP87 to form acetyl-CoA and carboxyholoBCCP87. The K(m) for holoBCCP87 was 0.45 +/- 0.07 mM while the V(max) was 2031.8 +/- 231.0 min(-1). The V/K or catalytic efficiency of carboxyltransferase with holoBCCP87 as substrate is 2000-fold greater than with biotin as substrate.     

Acetyl coenzyme A carbosylase. Circular dichroism studies of Escherichia coli biotin carboxyl carrier protein.

The biotin carboxyl carrier protein (BCCP) component of Escherichia coli acetyl coenzyme A carboxylase and three peptides derived from BCCP by proteolytic digestion have been examined by circular dichroism spectroscopy. BCCP, which has a peptide molecular weight of 22,500, has a spectrum typical of globular proteins with negative extrema at 222 nm and 208 nm. The two smallest peptides, BCCP(SC) and BCCP(9,100), with molecular weights of 8,900 and 9,100, respectively, exhibit unusual positive CD bands centered at 237 nm and 220 nm. BCCP(10,400), with a molecular weight of 10,400, has a CD spectrum intermediate between BCCP and that of the smallest peptides. Since d-biotin exhibits a positive CD band at 233 nm, it was suspected that the biotin prosthetic group might be the chromophore responsible for the 237 nm CD band seen in BCCP(SC) and BCCP(9,100). Enzymatic carboxylation of BCCP(SC) to form CO2-BCCP(SC) caused the CD spectrum to change with a shift of the 237 nm band to 232 nm. The positive CD band at 220 nm was unaffected by carboxylation of the biotin prosthetic group. These date suggest that the 237 nm signal may be due either to the biotin which acts as a chromophore directly or to a chromophore that is perturbed by the carboxylation of biotin. A spectropolarimetric titration was carried out to investigate the possible contribution of the single tyrosine residue of BCCP(SC) to the CD spectrum of this peptide. At pH values over 9 the CD spetrum changed with the disappearance of the 237 nm band, suggesting that tyrosine might contribute to this CD band. Denaturation of BCCP(SC) or BCCP(9,100) with 8 M urea of 6 M guanidine HCl abolished the positive CD bands and resulted in spectra typical of a random coil, whereas treatment of BCCP(SC) with 1% sodium dodecyl sulfate abolished the positive bands and left a spectrum exhibiting a shoulder at 222 nm and a negative band at 205 nm, suggestive of a high degree of ordered structure. It is concluded that the CD band at 237 nm in BCCP(SC) and BCCP(9,100) is prabably due to a noncovalent interaction of biotin with an amino acid residue(s) of the protein. It is suggested that the biotin prosthetic group is partially buried in the surface of the protein, rather than swinging free at the end of the lysine side chain through which it is covalently linked to the protein, to permit this interaction to occur.     

Nonenzymatic biotinylation of a biotin carboxyl carrier protein: unusual reactivity of the physiological target lysine

Enzyme-catalyzed addition of biotin to proteins is highly specific. In any single organism one or a small number of proteins are biotinylated and only a single lysine on each of these proteins is modified. A detailed understanding of the structural basis for the selective biotinylation process has not yet been elucidated. Recently certain mutants of the Escherichia coli biotin protein ligase have been shown to mediate "promiscuous" biotinylation of proteins. It was suggested that the reaction involved diffusion of a reactive activated biotin intermediate, biotinoyl-5'-AMP, with nonspecific proteins. In this work the reactivity of this chemically synthesized intermediate toward the natural target of enzymatic biotinylation, the biotin carboxyl carrier protein, was investigated. The results indicate that the intermediate does, indeed, react with target protein, albeit at a significantly slower rate than the enzyme-catalyzed process. Surprisingly, analysis of the products of nonenzymatic biotinylation indicates that of five lysine residues in the protein only the physiological target side chain is modified. These results indicate that either the environment of this lysine residue or its intrinsic properties render it highly reactive to nonenzymatic biotinylation mediated by biotinoyl-5'-AMP. This reactivity may be important for its selective biotinylation in vivo.     

The solution structure of acyl carrier protein from Mycobacterium tuberculosis

Acyl carrier protein (ACP) performs the essential function of shuttling the intermediates between the enzymes that constitute the type II fatty acid synthase system. Mycobacterium tuberculosis is unique in producing extremely long mycolic acids, and tubercular ACP, AcpM, is also unique in possessing a longer carboxyl terminus than other ACPs. We determined the solution structure of AcpM using protein NMR spectroscopy to define the similarities and differences between AcpM and the typical structures. The amino-terminal region of the structure is well defined and consists of four helices arranged in a right-handed bundle held together by interhelical hydrophobic interactions similar to the structures of other bacterial ACPs. The unique carboxyl-terminal extension from helix IV has a "melted down" feature, and the end of the molecule is a random coil. A comparison of the apo- and holo-forms of AcpM revealed that the 4'-phosphopantetheine group oscillates between two states; in one it is bound to a hydrophobic groove on the surface of AcpM, and in another it is solvent-exposed. The similarity between AcpM and other ACPs reveals the conserved structural motif that is recognized by all type II enzymes. However, the function of the coil domain extending from helix IV to the carboxyl terminus remains enigmatic, but its structural characteristics suggest that it may interact with the very long chain intermediates in mycolic acid biosynthesis or control specific protein-protein interactions.     

Saccharopolyspora hirsuta 367 encodes clustered genes similar to ketoacyl synthase, ketoacyl reductase, acyl carrier protein, and biotin carboxyl carrier protein

The actI gene, encoding a component of the actinorhodin polyketide synthase of Streptomyces coelicolor, was used to identify and clone a homologous 11.7 kb BamHI DNA fragment from Saccharopolyspora hirsuta 367. The cloned fragment complemented actinorhodin production in a strain of Streptomyces coelicolor bearing a mutant actI gene. The DNA sequence of a 5.1 kb fragment revealed 6 open reading frames (ORF). ORF1 does not resemble any known DNA or deduced protein sequence, while the deduced protein sequence of ORF2 resembles that of biotin carboxyl carrier proteins. Based on the similarity to deduced protein sequences from cloned genes of polyketide producers, ORF3 would code for a ketoreductase, ORF4 and ORF5 for the putative heterodimeric -ketoacyl synthase, and ORF6 for an acyl carrier protein.     

Enzymes for purine synthesis and scavenging in pathogenic mycobacteria and their distribution in Mycobacterium leprae

Three enzymes catalysing the synthesis of four intermediates (phosphoribosylglycinamide, phosphoribosylaminoimidazole-succinocarboxamide, phosphoribosylaminoimidazole-carboxamide and AMP) in the purine biosynthetic pathway were detected in extracts of Mycobacterium microti and M. avium, even when the organisms had been grown in mice. However only one of the three enzymes, adenylosuccinate AMP-lyase (catalysing the synthesis of the last two of the four intermediates listed above) was detected in M. leprae. Phosphoribosyltransferases, which convert adenine, guanine and hypoxanthine to the corresponding nucleoside monophosphates, and adenosine kinase were the major enzymes for purine scavenging in all mycobacteria studied. In contrast to enzymes in the synthetic pathway, evidence for metabolic regulation of the purine-scavenging enzymes was obtained. In particular, 20-80-fold differences in the activities of guanine phosphoribosyltransferase and adenosine kinase were observed when M. microti was grown in media with or without purines, or in mice. In M. leprae, activities of all phosphoribosyltransferases were low in comparison with activities in M. microti and M. avium (specific activity less than 2% when comparisons were made between extracts of host-grown mycobacteria). However, activity of adenosine kinase was higher in host-grown M. leprae than in host-grown M. microti or M. avium.     

Expression and characterization of the protein Rv1399c from Mycobacterium tuberculosis. A novel carboxyl esterase structurally related to the HSL family.

The Mycobacterium tuberculosis genome contains an unusually high number of proteins involved in the metabolism of lipids belonging to the Lip family, including various nonlipolytic and lipolytic hydrolases. Driven by a structural genomic approach, we have biochemically characterized the Rv1399c gene product, LipH, previously annotated as a putative lipase. Rv1399c was overexpressed in E. coli as inclusion bodies and refolded. Rv1399c efficiently hydrolyzes soluble triacylglycerols and vinyl esters. It is inactive against emulsified substrate and its catalytic activity is strongly inhibited by the diethyl paranitrophenyl phosphate (E600). These kinetic behaviors unambiguously classify Rv1399c as a nonlipolytic rather than a lipolytic hydrolase. Sequence alignment reveals that this enzyme belongs to the alpha/beta hydrolase fold family and shares 30-40% amino acid sequence identity with members of the hormone-sensitive lipase subfamily. A model of Rv1399c derived from homologous three-dimensional structures reveals a canonical catalytic triad (Ser162, His290 and Asp260) located at the bottom of a solvent accessible pocket lined by neutral or charged residues. Based on this model, kinetic data of the Arg213Ala mutant partially explain the role of the guanidinium moiety, located close to His290, to confer an unusual low pH shift of the catalytic histidine in the wild type enzyme. Overall, these data identify Rv1399c as a new nonlipolytic hydrolase from M. tuberculosis and we thus propose to reannotate its gene product as NLH-H.