Evolving the naturally compromised chorismate mutase from Mycobacterium tuberculosis to top performance

Chorismate mutase (CM), an essential enzyme at the branch-point of the shikimate pathway, is required for the biosynthesis of phenylalanine and tyrosine in bacteria, archaea, plants, and fungi. MtCM, the CM from Mycobacterium tuberculosis, has less than 1% of the catalytic efficiency of a typical natural CM and requires complex formation with 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase for high activity. To explore the full potential of MtCM for catalyzing its native reaction, we applied diverse iterative cycles of mutagenesis and selection, thereby raising kcat/Km 270-fold to 5 × 105m−1s−1, which is even higher than for the complex. Moreover, the evolutionarily optimized autonomous MtCM, which had 11 of its 90 amino acids exchanged, was stabilized compared with its progenitor, as indicated by a 9 °C increase in melting temperature. The 1.5 Å crystal structure of the top-evolved MtCM variant reveals the molecular underpinnings of this activity boost. Some acquired residues (e.g. Pro52 and Asp55) are conserved in naturally efficient CMs, but most of them lie beyond the active site. Our evolutionary trajectories reached a plateau at the level of the best natural enzymes, suggesting that we have exhausted the potential of MtCM. Taken together, these findings show that the scaffold of MtCM, which naturally evolved for mediocrity to enable inter-enzyme allosteric regulation of the shikimate pathway, is inherently capable of high activity.

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Biochemical and Structural Characterization of the Secreted Chorismate Mutase (Rv1885c) from Mycobacterium tuberculosis H37Rv: an *AroQ Enzyme Not Regulated by the Aromatic Amino Acids

Journal of Bacteriology ◽

10.1128/jb.00441-06 ◽

2006 ◽

Vol 188 (24) ◽

pp. 8638-8648 ◽

Cited By ~ 32

Author(s):

Sook-Kyung Kim ◽

Sathyavelu K. Reddy ◽

Bryant C. Nelson ◽

Gregory B. Vasquez ◽

Andrew Davis ◽

...

Keyword(s):

Amino Acids ◽

Mycobacterium Tuberculosis ◽

Active Site ◽

Signal Sequence ◽

Aromatic Amino Acids ◽

Helical Structure ◽

Data Bank ◽

Chorismate Mutase ◽

Site Directed Mutagenesis ◽

Single Chain

ABSTRACT The gene Rv1885c from the genome of Mycobacterium tuberculosis H37Rv encodes a monofunctional and secreted chorismate mutase (*MtCM) with a 33-amino-acid cleavable signal sequence; hence, it belongs to the *AroQ class of chorismate mutases. Consistent with the heterologously expressed *MtCM having periplasmic destination in Escherichia coli and the absence of a discrete periplasmic compartment in M. tuberculosis, we show here that *MtCM secretes into the culture filtrate of M. tuberculosis. *MtCM functions as a homodimer and exhibits a dimeric state of the protein at a concentration as low as 5 nM. *MtCM exhibits simple Michaelis-Menten kinetics with a Km of 0.5 ± 0.05 mM and a k cat of 60 s−1 per active site (at 37°C and pH 7.5). The crystal structure of *MtCM has been determined at 1.7 Å resolution (Protein Data Bank identifier 2F6L). The protein has an all alpha-helical structure, and the active site is formed within a single chain without any contribution from the second chain in the dimer. Analysis of the structure shows a novel fold topology for the protein with a topologically rearranged helix containing Arg134. We provide evidence by site-directed mutagenesis that the residues Arg49, Lys60, Arg72, Thr105, Glu109, and Arg134 constitute the catalytic site; the numbering of the residues includes the signal sequence. Our investigation on the effect of phenylalanine, tyrosine, and tryptophan on *MtCM shows that *MtCM is not regulated by the aromatic amino acids. Consistent with this observation, the X-ray structure of *MtCM does not have an allosteric regulatory site.

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Reconstruction of Mycobacterial Dehalogenase Rv2579 by Cumulative Mutagenesis of Haloalkane Dehalogenase LinB

Applied and Environmental Microbiology ◽

10.1128/aem.69.4.2349-2355.2003 ◽

2003 ◽

Vol 69 (4) ◽

pp. 2349-2355 ◽

Cited By ~ 18

Author(s):

Yuji Nagata ◽

Zbyněk Prokop ◽

Soňa Marvanová ◽

Jana Sýkorová ◽

Marta Monincová ◽

...

Keyword(s):

Crystal Structure ◽

Mycobacterium Tuberculosis ◽

Amino Acid ◽

Active Site ◽

Homology Model ◽

Protein Family ◽

Sphingomonas Paucimobilis ◽

Amino Acid Residues ◽

Haloalkane Dehalogenase

ABSTRACT The homology model of protein Rv2579 from Mycobacterium tuberculosis H37Rv was compared with the crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26, and this analysis revealed that 6 of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. To characterize the effect of replacement of these six amino acid residues, mutations were introduced cumulatively into the six amino acid residues of LinB. The sixfold mutant, which was supposed to have the active site of Rv2579, exhibited haloalkane dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase protein family.

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Potent Inhibitors of a Shikimate Pathway Enzyme from Mycobacterium tuberculosis

Journal of Biological Chemistry ◽

10.1074/jbc.m110.211649 ◽

2011 ◽

Vol 286 (18) ◽

pp. 16197-16207 ◽

Cited By ~ 26

Author(s):

Sebastian Reichau ◽

Wanting Jiao ◽

Scott R. Walker ◽

Richard D. Hutton ◽

Edward N. Baker ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Conformational Changes ◽

Active Site ◽

Catalytic Mechanism ◽

Condensation Reaction ◽

Shikimate Pathway ◽

Enzyme Reaction ◽

Multidrug Resistant ◽

Active Site Residues ◽

Site Water

Tuberculosis remains a serious global health threat, with the emergence of multidrug-resistant strains highlighting the urgent need for novel antituberculosis drugs. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the shikimate pathway for the biosynthesis of aromatic compounds. This pathway has been shown to be essential in Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. DAH7PS catalyzes a condensation reaction between P-enolpyruvate and erythrose 4-phosphate to give 3-deoxy-d-arabino-heptulosonate 7-phosphate. The enzyme reaction mechanism is proposed to include a tetrahedral intermediate, which is formed by attack of an active site water on the central carbon of P-enolpyruvate during the course of the reaction. Molecular modeling of this intermediate into the active site reported in this study shows a configurational preference consistent with water attack from the re face of P-enolpyruvate. Based on this model, we designed and synthesized an inhibitor of DAH7PS that mimics this reaction intermediate. Both enantiomers of this intermediate mimic were potent inhibitors of M. tuberculosis DAH7PS, with inhibitory constants in the nanomolar range. The crystal structure of the DAH7PS-inhibitor complex was solved to 2.35 Å. Both the position of the inhibitor and the conformational changes of active site residues observed in this structure correspond closely to the predictions from the intermediate modeling. This structure also identifies a water molecule that is located in the appropriate position to attack the re face of P-enolpyruvate during the course of the reaction, allowing the catalytic mechanism for this enzyme to be clearly defined.

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Crystal Structure of Mycobacterium tuberculosis Polyketide Synthase 11 (PKS11) Reveals Intermediates in the Synthesis of Methyl-branched Alkylpyrones

Journal of Biological Chemistry ◽

10.1074/jbc.m113.468892 ◽

2013 ◽

Vol 288 (23) ◽

pp. 16484-16494 ◽

Cited By ~ 17

Author(s):

Kuppan Gokulan ◽

Seán E. O'Leary ◽

William K. Russell ◽

David H. Russell ◽

Mallikarjun Lalgondar ◽

...

Keyword(s):

Crystal Structure ◽

Mycobacterium Tuberculosis ◽

Active Site ◽

Polyketide Synthase ◽

Biological Function ◽

Type Iii ◽

Optimal Activity ◽

Malonyl Coa ◽

Hydrophobic Tunnel ◽

Reaction In Solution

PKS11 is one of three type III polyketide synthases (PKSs) identified in Mycobacterium tuberculosis. Although many PKSs in M. tuberculosis have been implicated in producing complex cell wall glycolipids, the biological function of PKS11 is unknown. PKS11 has previously been proposed to synthesize alkylpyrones from fatty acid substrates. We solved the crystal structure of M. tuberculosis PKS11 and found the overall fold to be similar to other type III PKSs. PKS11 has a deep hydrophobic tunnel proximal to the active site Cys-138 to accommodate substrates. We observed electron density in this tunnel from a co-purified molecule that was identified by mass spectrometry to be palmitate. Co-crystallization with malonyl-CoA (MCoA) or methylmalonyl-CoA (MMCoA) led to partial turnover of the substrate, resulting in trapped intermediates. Reconstitution of the reaction in solution confirmed that both co-factors are required for optimal activity, and kinetic analysis shows that MMCoA is incorporated first, then MCoA, followed by lactonization to produce methyl-branched alkylpyrones.

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Crystal structure of chorismate mutase fromBurkholderia phymatum

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x18002868 ◽

2018 ◽

Vol 74 (4) ◽

pp. 187-192 ◽

Cited By ~ 1

Author(s):

Oluwatoyin A. Asojo ◽

Sandhya Subramanian ◽

Jan Abendroth ◽

Ilyssa Exley ◽

Donald D. Lorimer ◽

...

Keyword(s):

Crystal Structure ◽

Drug Development ◽

Structural Genomics ◽

Active Site ◽

Chorismate Mutase ◽

Biological Warfare ◽

Beneficial Bacteria ◽

Sequence Identity ◽

Resolution Structure

The bacteriumBurkholderia phymatumis a promiscuous symbiotic nitrogen-fixating bacterium that belongs to one of the largest groups of Betaproteobacteria. OtherBurkholderiaspecies are known to cause disease in plants and animals, and some are potential agents for biological warfare. Structural genomics efforts include characterizing the structures of enzymes from pathways that can be targeted for drug development. As part of these efforts, chorismate mutase fromB. phymatumwas produced and crystallized, and a 1.95 Å resolution structure is reported. This enzyme shares less than 33% sequence identity with other homologs of known structure. There are two classes of chorismate mutase: AroQ and AroH. The bacterial subclass AroQγ has reported roles in virulence. Chorismate mutase fromB. phymatumhas the prototypical AroQγ topology and retains the characteristic chorismate mutase active site. This suggests that substrate-based chorismate mutase inhibitors will not be specific and are likely to affect beneficial bacteria such asB. phymatum.

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Determinants of specificity for aflatoxin B1-8,9-epoxide in Alpha-class glutathione S-transferases

Biochemical Journal ◽

10.1042/bj3390095 ◽

1999 ◽

Vol 339 (1) ◽

pp. 95-101 ◽

Cited By ~ 4

Author(s):

Paul D. McDONAGH ◽

David J. JUDAH ◽

John D. HAYES ◽

Lu-Yun LIAN ◽

Gordon E. NEAL ◽

...

Keyword(s):

Crystal Structure ◽

Substrate Specificity ◽

High Activity ◽

Aflatoxin B1 ◽

Active Site ◽

Homology Modelling ◽

Three Dimensional ◽

Glutathione S Transferase ◽

Glutathione S Transferases ◽

Aspartate Residue

We have used homology modelling, based on the crystal structure of the human glutathione S-transferase (GST) A1-1, to obtain the three-dimensional structures of rat GSTA3 and rat GSTA5 subunits bound to S-aflatoxinyl–glutathione. The resulting models highlight two residues, at positions 208 and 108, that could be important for determining, either directly or indirectly, substrate specificity for aflatoxin-exo-8,9-epoxide among the Alpha-class GSTs. Residues at these positions were mutated in human GSTA1-1 (Met-208, Leu-108), rat GSTA3-3 (Glu-208, His-108) and rat GSTA5-5 (Asp-208, Tyr-108): in the active rat GSTA5-5 to those in the inactive GSTA1-1; and in the inactive human GSTA1-1 and rat GSTA3-3 to those in the active rat GSTA5-5. These studies show clearly that, in all three GSTs, an aspartate residue at position 208 is a prerequisite for high activity in aflatoxin-exo-8,9-epoxide conjugation, although this alone is not sufficient; other residues in the vicinity, particularly residues 103–112, are important, perhaps for the optimal orientation of the aflatoxin-exo-8,9-epoxide in the active site for catalysis to occur.

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Regulation of the intersubunit ammonia tunnel in Mycobacterium tuberculosis glutamine-dependent NAD+ synthetase

Biochemical Journal ◽

10.1042/bj20112210 ◽

2012 ◽

Vol 443 (2) ◽

pp. 417-426 ◽

Cited By ~ 10

Author(s):

Watchalee Chuenchor ◽

Tzanko I. Doukov ◽

Melissa Resto ◽

Andrew Chang ◽

Barbara Gerratana

Keyword(s):

Mycobacterium Tuberculosis ◽

Active Site ◽

Drug Target ◽

Wild Type ◽

Ammonia Transport ◽

Site Variation ◽

Ammonia Tunnel ◽

Essential Enzyme ◽

Nad Synthetase ◽

Insight Into

Glutamine-dependent NAD+ synthetase is an essential enzyme and a validated drug target in Mycobacterium tuberculosis (mtuNadE). It catalyses the ATP-dependent formation of NAD+ from NaAD+ (nicotinic acid–adenine dinucleotide) at the synthetase active site and glutamine hydrolysis at the glutaminase active site. An ammonia tunnel 40 Å (1 Å=0.1 nm) long allows transfer of ammonia from one active site to the other. The enzyme displays stringent kinetic synergism; however, its regulatory mechanism is unclear. In the present paper, we report the structures of the inactive glutaminase C176A variant in an apo form and in three synthetase–ligand complexes with substrates (NaAD+/ATP), substrate analogue {NaAD+/AMP-CPP (adenosine 5′-[α,β-methylene]triphosphate)} and intermediate analogues (NaAD+/AMP/PPi), as well as the structure of wild-type mtuNadE in a product complex (NAD+/AMP/PPi/glutamate). This series of structures provides snapshots of the ammonia tunnel during the catalytic cycle supported also by kinetics and mutagenesis studies. Three major constriction sites are observed in the tunnel: (i) at the entrance near the glutaminase active site; (ii) in the middle of the tunnel; and (iii) at the end near the synthetase active site. Variation in the number and radius of the tunnel constrictions is apparent in the crystal structures and is related to ligand binding at the synthetase domain. These results provide new insight into the regulation of ammonia transport in the intermolecular tunnel of mtuNadE.

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Crystal structure of Rv1220c, a SAM-dependentO-methyltransferase fromMycobacterium tuberculosis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x17006057 ◽

2017 ◽

Vol 73 (6) ◽

pp. 315-320 ◽

Cited By ~ 1

Author(s):

Qiaoling Yan ◽

Neil Shaw ◽

Lanfang Qian ◽

Dunquan Jiang

Keyword(s):

Crystal Structure ◽

Escherichia Coli ◽

Mycobacterium Tuberculosis ◽

Electron Density ◽

Active Site ◽

Metal Binding ◽

Structural Information

Rv1220c fromMycobacterium tuberculosisis annotated as anO-methyltransferase (MtbOMT). Currently, no structural information is available for this protein. Here, the crystal structure ofMtbOMT refined to 2.0 Å resolution is described. The structure reveals the presence of a methyltransferase fold and shows clear electron density for one molecule ofS-adenosylmethionine (SAM), which was apparently bound by the protein during its production inEscherichia coli. Although the overall structure ofMtbOMT resembles the structures ofO-methyltransferases fromCornybacterium glutamicum,Coxiella burnettiandAlfa alfa, differences are observed in the residues that make up the active site. Notably, substitution of Asp by His164 seems to abrogate metal binding byMtbOMT. A putative catalytic His–Asp pair located in the vicinity of SAM is absolutely conserved inMtbOMT homologues from all species ofMycobacterium, suggesting a conserved function for this protein.

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