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1.  Molecular Basis of E. coli L-Threonine Aldolase Catalytic Inactivation at Low pH 
Biochimica et biophysica acta  2015;1854(4):278-283.
L-Threonine aldolases (TAs), a family of enzymes belonging to the fold-type I pyridoxal 5′-phosphate (PLP) dependent enzymes, play a role in catalyzing the reversible cleavage of L-3-hydroxy-α-amino acids to glycine and the corresponding aldehydes. Threonine aldolases have great biotechnological potential for the syntheses of pharmaceutically relevant drug molecules because of their stereospecificity. The pH-dependency of their catalytic activity, affecting reaction intermediates, led us to study the effect of low-pH on E. coli TA (eTA) structure. We report here a low-pH crystal structure of eTA at 2.1 Å resolution, with a noncovalently bound uncleaved L-serine substrate, and a PLP cofactor bound as an internal aldimine. This structure contrasts with other eTA structures obtained at physiological pH that show products or substrates bound as PLP-external aldimines. The non-productive binding at low-pH is due to an unusual substrate serine binding orientation in which the α-amino group and carboxylate group are in the wrong positions (relative to the active site residues) as a result of protonation of the α-amino group of the serine, as well as the active site histidines, His83 and His126. Protonation of these residues prevent the characteristic nucleophilic attack of the α-amino group of substrate serine on C4′ of PLP to form the external aldimine. Our study shows that at low pH the change in charge distribution at the active site can result in substrates binding in a non-productive orientation.
PMCID: PMC4323617  PMID: 25560296
pyridoxal 5′-phosphate; threonine aldolase; internal aldimine; transaldimination; PLP-dependent enzymes; protein crystallography
The FEBS journal  2013;281(1):129-145.
L-Threonine aldolases (TAs) represent a family of homologous pyridoxal 5’-phosphate-dependent enzymes found in bacteria and fungi, and catalyse the reversible cleavage of several l-3-hydroxy-α-amino acids. TAs have great biotechnological potential, since they catalyse the formation of carbon-carbon bonds, and therefore may be exploited for bioorganic synthesis of l-3-hydroxyamino acids that are biologically active or constitute building blocks for pharmaceutical molecules. Many TAs, showing different stereospecificity towards the Cβ configuration, have been isolated. Because of their potential to carry out diastereoselective syntheses, TAs have been the subject of structural, functional and mechanistic studies. Nevertheless, their catalytic mechanism and the structural bases of their stereospecificity have not been elucidated.
In this study, we have determined the crystal structure of low-specificity l-threonine aldolase from Escherichia coli at 2.2 Å resolution, in the unliganded form and co-crystallized with l-serine and l-threonine. Furthermore, several active-site mutants have been functionally characterised in order to elucidate the reaction mechanism and the molecular bases of stereospecificity. No active site catalytic residue was revealed, and a structural water molecule was assumed to act as catalytic base in the retro-aldol cleavage reaction.
Interestingly, the very large active site opening of E. coli TA suggests that a much larger molecule than l-threonine isomers may be easily accommodated, and threonine aldolases may actually play diverse physiological functions in different organisms. Substrate recognition and reaction specificity seem to be guided by the overall microenvironment that surrounds the substrate at the enzyme active site, rather than to one ore more specific residues.
PMCID: PMC4366684  PMID: 24165453
threonine aldolase structure; catalytic mechanism; substrate preference; catalytic water; protein crystallography
3.  Crystallographic analysis of human hemoglobin elucidates the structural basis of the potent and dual antisickling activity of pyridyl derivatives of vanillin. Corrigendum 
A correction to the paper by Abdulmalik et al. [(2011), Acta Cryst. D67, 920–928].
The affiliation of one of the authors of Abdulmalik et al. (2011) [Acta Cryst. D67, 920–928] is corrected.
PMCID: PMC3337008
hemoglobin; oxygen affinity; sickle-cell disease; polymerization; T state; R2 state; corrigendum
4.  Crystallographic analysis of human hemoglobin elucidates the structural basis of the potent and dual antisickling activity of pyridyl derivatives of vanillin 
Pyridyl derivatives of vanillin increase the fraction of the more soluble oxygenated sickle hemoglobin and/or directly increase the solubility of deoxygenated sickle hemoglobin. Crystallographic analysis reveals the structural basis of the potent and dual antisickling activity of these derivatives.
Vanillin has previously been studied clinically as an antisickling agent to treat sickle-cell disease. In vitro investigations with pyridyl derivatives of vanillin, including INN-312 and INN-298, showed as much as a 90-fold increase in antisickling activity compared with vanillin. The compounds preferentially bind to and modify sickle hemoglobin (Hb S) to increase the affinity of Hb for oxygen. INN-312 also led to a considerable increase in the solubility of deoxygenated Hb S under completely deoxygenated conditions. Crystallographic studies of normal human Hb with INN-312 and INN-298 showed that the compounds form Schiff-base adducts with the N-terminus of the α-subunits to constrain the liganded (or relaxed-state) Hb conformation relative to the unliganded (or tense-state) Hb conformation. Interestingly, while INN-298 binds and directs its meta-positioned pyridine-methoxy moiety (relative to the aldehyde moiety) further down the central water cavity of the protein, that of INN-312, which is ortho to the aldehyde, extends towards the surface of the protein. These studies suggest that these compounds may act to prevent sickling of SS cells by increasing the fraction of the soluble high-affinity Hb S and/or by stereospecific inhibition of deoxygenated Hb S polymerization.
PMCID: PMC3211971  PMID: 22101818
hemoglobin; oxygen affinity; sickle-cell disease; polymerization; T state; R2 state
5.  Pyridoxal 5′-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase 
PLoS ONE  2012;7(7):e41680.
Pyridoxal 5′-phosphate (PLP) is a cofactor for dozens of B6 requiring enzymes. PLP reacts with apo-B6 enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B6 enzyme. During protein turnover, the PLP is salvaged by first converting it to pyridoxal by a phosphatase and then back to PLP by pyridoxal kinase. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 4′-aldehyde moiety forms covalent adducts with other compounds and non-B6 proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complex with the product PLP to form an inactive enzyme complex. Evidence is presented that, in the inhibited complex, PLP has formed an aldimine bond with an active site lysine residue during catalytic turnover. The rate of dissociation of PLP from the complex is very slow, being only partially released after a 2-hour incubation with PLP phosphatase. Interestingly, the inactive pyridoxal kinase•PLP complex can be partially reactivated by transferring the tightly bound PLP to an apo-B6 enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell.
PMCID: PMC3404986  PMID: 22848564
6.  Crystal Structures of Human Pyridoxal Kinase in Complex with the Neurotoxins, Ginkgotoxin and Theophylline: Insights into Pyridoxal Kinase Inhibition 
PLoS ONE  2012;7(7):e40954.
Several drugs and natural compounds are known to be highly neurotoxic, triggering epileptic convulsions or seizures, and causing headaches, agitations, as well as other neuronal symptoms. The neurotoxic effects of some of these compounds, including theophylline and ginkgotoxin, have been traced to their inhibitory activity against human pyridoxal kinase (hPL kinase), resulting in deficiency of the active cofactor form of vitamin B6, pyridoxal 5′-phosphate (PLP). Pyridoxal (PL), an inactive form of vitamin B6 is converted to PLP by PL kinase. PLP is the B6 vitamer required as a cofactor for over 160 enzymatic activities essential in primary and secondary metabolism. We have performed structural and kinetic studies on hPL kinase with several potential inhibitors, including ginkgotoxin and theophylline. The structural studies show ginkgotoxin and theophylline bound at the substrate site, and are involved in similar protein interactions as the natural substrate, PL. Interestingly, the phosphorylated product of ginkgotoxin is also observed bound at the active site. This work provides insights into the molecular basis of hPL kinase inhibition and may provide a working hypothesis to quickly screen or identify neurotoxic drugs as potential hPL kinase inhibitors. Such adverse effects may be prevented by administration of an appropriate form of vitamin B6, or provide clues of how to modify these drugs to help reduce their hPL kinase inhibitory effects.
PMCID: PMC3412620  PMID: 22879864
7.  Application of a Newly Identified and Characterized 18-O-Acyltransferase in Chemoenzymatic Synthesis of Selected Natural and Nonnatural Bioactive Derivatives of Phoslactomycins▿  
Applied and Environmental Microbiology  2009;75(11):3469-3476.
Phoslactomycins (PLMs) and related leustroducsins (LSNs) have been isolated from a variety of bacteria based on antifungal, anticancer, and other biological assays. Streptomyces sp. strain HK 803 produces five PLM analogs (PLM A and PLMs C to F) in which the C-18 hydroxyl substituent is esterified with a range of branched, short-alkyl-chain carboxylic acids. The proposed pathway intermediate, PLM G, in which the hydroxyl residue is not esterified has not been observed at any significant level in fermentation, and the only route to this potentially useful intermediate has been an enzymatic deacylation of other PLMs and LSNs. We report that deletion of plmS3 from the PLM biosynthetic cluster gives rise to a mutant which accumulates the PLM G intermediate. The 921-bp plmS3 open reading frame was cloned and expressed as an N-terminally polyhistidine-tagged protein in Escherichia coli and shown to be an 18-O acyltransferase, catalyzing conversion of PLM G to PLM A, PLM C, and PLM E using isobutyryl coenzyme A (CoA), 3-methylbutyryl-CoA, and cyclohexylcarbonyl-CoA, respectively. The efficiency of this process (kcat of 28 ± 3 min−1 and Km of 88 ± 16 μM) represents a one-step chemoenzymatic alternative to a multistep synthetic process for selective chemical esterification of the C-18 hydroxy residue of PLM G. PlmS3 was shown to catalyze esterification of PLM G with CoA and N-acetylcysteamine thioesters of various saturated, unsaturated, and aromatic carboxylic acids and thus also to provide an efficient chemoenzymatic route to new PLM analogs.
PMCID: PMC2687304  PMID: 19304832
8.  The plmS2-Encoded Cytochrome P450 Monooxygenase Mediates Hydroxylation of Phoslactomycin B in Streptomyces sp. Strain HK803 
Journal of Bacteriology  2005;187(23):7970-7976.
Streptomyces sp. strain HK803 produces six analogues of phoslactomycin (Plm A through Plm F). With the exception of Plm B, these analogues contain a C-18 hydroxyl substituent esterified with a range of short-alkyl-chain carboxylic acids. Deletion of the plmS2 open reading frame (ORF), showing high sequence similarity to bacterial cytochrome P450 monooxygenases (CYPs), from the Plm biosynthetic gene cluster has previously resulted in an NP1 mutant producing only Plm B (N. Palaniappan, B. S. Kim, Y. Sekiyama, H. Osada, and K. A. Reynolds, J. Biol. Chem. 278:35552-35557, 2003). Herein, we report that a complementation experiment with an NP1 derivative (NP2), using a recombinant conjugative plasmid carrying the plmS2 ORF downstream of the ermE* constitutive promoter (pMSG1), restored production of Plm A and Plm C through Plm F. The 1.2-kbp plmS2 ORF was also expressed efficiently as an N-terminal polyhistidine-tagged protein in Streptomyces coelicolor. The recombinant PlmS2 converted Plm B to C-18-hydroxy Plm B (Plm G). PlmS2 was highly specific for Plm B and unable to process a series of derivatives in which either the lactone ring was hydrolyzed or the C-9 phosphate ester was converted to C-9/C-11 phosphorinane. This biochemical analysis and complementation experiment are consistent with a proposed Plm biosynthetic pathway in which the penultimate step is hydroxylation of the cyclohexanecarboxylic acid-derived side chain of Plm B by PlmS2 (the resulting Plm G is then esterified to provide Plm A and Plm C through Plm F). Kinetic parameters for Plm B hydroxylation by PlmS2 (Km of 45.3 ± 9.0 μM and kcat of 0.27 ± 0.04 s−1) are consistent with this step being a rate-limiting step in the biosynthetic pathway. The penultimate pathway intermediate Plm G has less antifungal activity than Plm A through Plm F and is not observed in fermentations of either the wild-type strain or NP2/pMSG1.
PMCID: PMC1291264  PMID: 16291670
9.  Molecular and Biotechnological Aspects of Microbial Proteases† 
Proteases represent the class of enzymes which occupy a pivotal position with respect to their physiological roles as well as their commercial applications. They perform both degradative and synthetic functions. Since they are physiologically necessary for living organisms, proteases occur ubiquitously in a wide diversity of sources such as plants, animals, and microorganisms. Microbes are an attractive source of proteases owing to the limited space required for their cultivation and their ready susceptibility to genetic manipulation. Proteases are divided into exo- and endopeptidases based on their action at or away from the termini, respectively. They are also classified as serine proteases, aspartic proteases, cysteine proteases, and metalloproteases depending on the nature of the functional group at the active site. Proteases play a critical role in many physiological and pathophysiological processes. Based on their classification, four different types of catalytic mechanisms are operative. Proteases find extensive applications in the food and dairy industries. Alkaline proteases hold a great potential for application in the detergent and leather industries due to the increasing trend to develop environmentally friendly technologies. There is a renaissance of interest in using proteolytic enzymes as targets for developing therapeutic agents. Protease genes from several bacteria, fungi, and viruses have been cloned and sequenced with the prime aims of (i) overproduction of the enzyme by gene amplification, (ii) delineation of the role of the enzyme in pathogenecity, and (iii) alteration in enzyme properties to suit its commercial application. Protein engineering techniques have been exploited to obtain proteases which show unique specificity and/or enhanced stability at high temperature or pH or in the presence of detergents and to understand the structure-function relationships of the enzyme. Protein sequences of acidic, alkaline, and neutral proteases from diverse origins have been analyzed with the aim of studying their evolutionary relationships. Despite the extensive research on several aspects of proteases, there is a paucity of knowledge about the roles that govern the diverse specificity of these enzymes. Deciphering these secrets would enable us to exploit proteases for their applications in biotechnology.
PMCID: PMC98927  PMID: 9729602

Results 1-9 (9)