Qamar Bashir

Highly Thermostable l-Threonine Dehydrogenase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis

l-Threonine dehydrogenase, a key enzyme in the l-threonine metabolism, catalyses the NAD(+)-dependent conversion of l-threonine to 2-amino-3-ketobutyrate, that non-enzymically decarboxylates to aminoacetone. A search of the genome sequence of hyperthermophilic archaeon, Thermococcus kodakaraensis revealed the presence of a closely related orthologue (TK0916) of archaeal and bacterial l-threonine dehydrogenase genes. Expression in Escherichia coli, purification and characterization of the TK0916 gene product revealed that this gene actually coded for a protein with high levels of l-threonine dehydrogenase activity (7.26 U mg(-1)). The enzyme exhibited highest activity at pH 12 and 90 degrees C. The K(m) values for l-threonine and NAD(+) at 50 degrees C were 1.6 mM and 0.028 mM, respectively. The enzyme activity was dependent on divalent cations. The half-life of the enzyme was more than 2 h at 85 degrees C and 24 min in boiling water. This is the most thermostable threonine dehydrogenase exhibiting optimal activity at the highest pH (12) reported to date. This is the first report on the characterization of a TDH from genus Thermococcus.

Mechanistic and stereochemical studies of glycine oxidase from Bacillus subtilis strain R5.

Glycine oxidase gene from a strain of Bacillus subtilis was cloned and expressed in Escherichia coli. The purified enzyme was found, by mass spectrometry, to have a protein M(r) of 40763 (value of 40761.6 predicted from DNA sequence) and a FAD prosthetic group M(r) of 785.1 (theoretical value of 785.5). Glycine oxidase optimally catalyzes the conversion of glycine and oxygen into glyoxylate, hydrogen peroxide, and ammonia. Using samples of [2-RS-(3)H(2),2-(14)C]-, [2-R-(3)H,2-(14)C]-, and [2-S-(3)H,2-(14)C]glycine, we found that in the overall process H(Si) is removed. Incubation of the enzyme with [2-RS-(3)H(2),2-(14)C]glycine under anaerobic conditions, when only the reducing half of the reaction can occur, led to the recovery of 98.5% of the original glycine, which had the same (3)H:(14)C ratio as the starting substrate. The primary isotope effect was studied using [2-(2)H(2)]glycine, and we found that the specificity constants, k(cat)/K(M), for the protio and deuterio substrates were 1.46 x 10(3) and 1.05 x 10(2) M(-1) s(-1), respectively. Two alternative mechanisms for FAD-containing oxidases that involve either the intermediacy of a FADH(2)-imino acid complex or an amino acid covalently linked to FAD, formed via a carbanion, have been considered. The current knowledge of the mechanisms is reviewed, and we argue that a mechanism involving the FADH(2)-imino acid complex can be dissected to satisfactorily explain some of puzzling observations for which the carbanion mechanism was originally conceived. Furthermore, our results, together with observations in the literature, suggest that the interaction of glycine with the enzyme occurs within a tight ternary complex, which is protected from the protons of the medium.

TK1299, a highly thermostable NAD(P)H oxidase from Thermococcus kodakaraensis exhibiting higher enzymatic activity with NADPH.

Seven nicotinamide adenine dinucleotide oxidase homologs have been found in the genome of Thermococcus kodakaraensis. The gene encoding one of them, TK1299, consisted of 1326 nucleotides, corresponding to a polypeptide of 442 amino acids. To examine the molecular properties of TK1299, the structural gene was cloned, expressed in Escherichia coli and the gene product was characterized. Molecular weight of the recombinant protein was 49,375 Da when determined by matrix-assisted laser desorption/ionization time-of-flight and 300 kDa when analyzed by gel filtration chromatography indicating that it existed in a hexameric form. The enzyme was highly thermostable even in boiling water where it exhibited more than 95% of the enzyme activity after incubation of 150 min. TK1299 catalyzed the oxidation of NADH as well as NADPH and predominantly converted O₂ to H₂O (more than 75%). K(m) value of the enzyme towards NADH and NADPH was almost same (24 ± 2 μM) where as specific activity was higher with NADPH compared to NADH. To our knowledge this is the most thermostable and unique NAD(P)H oxidase displaying higher enzyme activity with NADPH.

Mechanism and substrate stereochemistry of 2-amino-3-oxobutyrate CoA ligase: implications for 5-aminolevulinate synthase and related enzymes

The condensation process catalysed by 2-amino-3-oxobutyrate CoA ligase (KBL; also known as 2-amino-3-ketobutyrate ligase) involves the loss of the pro-R hydrogen atom of glycine and occurs with the inversion of stereochemistry; a similar scenario is envisaged for the condensation step of other alpha-oxoamine synthases.

Genetic polymorphism among five species of Indus River carps on the basis of random amplified polymorphic DNA (RAPD) analysis

The knowledge of molecular genetics is a key to understand biodiversity, evolutionary relationship, conservation programs, and genetic structure of species of an ecosystem. To study genetic polymorphism in morphological variants of five species of Indus River carps, namely, Labeo gonius, Labeo rohita, Labeo calbasu, Catla catla, and Cirrhinus mrigala, random amplified polymorphic DNA (RAPD) analysis was used. Using eight primers, a total of 719 loci were amplified comprising of 389 polymorphic loci (53.7%) and 330 monomorphic loci (45.3%). The mean value of observed number of alleles (na), effective number of alleles (ne), Nei gene diversity (h), and Shannon’s information index were 2, 1.65, 0.38, and 0.56, respectively. The mean value of Nei total gene diversity (Ht), gene diversity within the population (Hs), relative magnitude of differentiation among population (Gst), and gene flow (Nm) were 0.38, 0.26, 0.28, and 3.98, respectively. The mean value of Hs and Gst suggests moderate gene flow between these fish populations. This study revealed that C. catla is more close to L. gonius, L. rohita, and L. calbasu than C. mrigala. We have presented the first detailed study of genetic variation within and among five fish species of Indus River carps in Pakistan.

TK1656, an L-asparaginase from Thermococcus kodakarensis, a novel candidate for therapeutic applications

Abstract L-Asparaginase is a potential therapeutic agent owing to its anti-tumor activity. We have previously characterized a thermostable L-asparaginase (TK1656F from Thermococcus kodakaraensis that exhibits highest ever reported enzymatic activity, no glutaminase activity and high stability. In this study we have compared the cytotoxicity and anti-proliferative activity of TK1656 and a commercially available L-asparaginase from Escherichia coli on cancerous cells. Despite of the thermophilic origin, the amount of TK1656 required to inhibit equivalent growth of HeLa cells at 37°C was three times less than that of the L-asparaginase from E. coli. It is worth mentioning that TK1656 showed much reduced cytotoxic effect on non-cancerous cells as compared to the E. coli counterpart. Furthermore, TK1656 was found to potentiate induction of apoptosis of cancerous cells. Overall, our findings suggest that TK1656 harbors a remarkable potential for the cancer chemotherapeutics.