Methylglyoxal Binding to Bovine Liver Catalase Results in Loss of Activity and Heme Dislocation

Glycation, the non-enzymatic attachment of glucose to protein, is one of the important events in the pathophysiology of diabetes mellitus, Alzheimer’s, Parkinson’s and other diseases. Methylglyoxal (MG), a dicarbonyl compound formed during glycation, monosaccharide autoxidation, and metabolism is elevated during diabetes mellitus. Among other antioxidant enzymes, catalase is important for the defense against oxidative damage. However, antioxidant enzymes including catalase can themselves become targets of non-enzymatic modification by methylglyoxal. In this study, catalase was incubated with increasing concentrations of MG for different time intervals. Structural and functional alterations to catalase were monitored by a variety of approaches, namely, assay of enzyme activity, staining of gels for activity as well as heme, measurement of protein carbonyls and Arg pyrimidine, which is a specific MG modification product. A progressive increase in electrophoretic mobility and detachment of heme from the monomer were observed with increasing concentrations of methylglyoxal. The MG-modified enzyme showed reduced affinity towards the substrate hydrogen peroxide. Molecular modeling studies revealed that MG can access the heme and arginine residues close to it. Thus, the decrease in activity of methylglyoxal-modified catalase may be important in aggravating the severity of secondary complications seen in diabetes mellitus.HIGHLIGHTS•Increase in concentration of methylglyoxal caused a progressive increase in electrophoretic mobility and detachment of heme from the monomer.•MG-modified enzyme showed reduced affinity towards the substrate hydrogen peroxide.•Molecular modeling studies showed that MG can access the heme and arginine residues close to it

Hydrolysis of Cyclic Ureas under Microwave Irradiation: Synthesis and Characterization of 7,8-Diaminopelargonic Acid

A simple and efficient method for the synthesis of 7,8-diaminopelargonic acid, a key intermediate in the biotin biosynthesis pathway, is reported. The d-desthiobiotin powder was dissolved in concentrated hydrochloric acid, and the solution was exposed to microwave radiation of 2.45 GHz for varying lengths of time ranging from 60 s to 2 min. The product thus obtained was characterized by spectroscopic techniques and confirmed through bioassay. Further, the protocol was extended to the synthesis of several diamines from their corresponding cyclic ureas. The results show that the method is generally applicable and not only accelerates the hydrolysis reaction but also offers excellent yields

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of pantothenate kinase from Mycobacterium tuberculosis

Pantothenate kinase, the first enzyme of the universal coenzyme A biosynthetic pathway, from M. tuberculosis H37Rv has been cloned, expressed, purified and X-ray analysed in two different crystal forms

Polymyxin B: an ode to an old antidote for endotoxic shock

Endotoxic shock, a syndrome characterized by deranged hemodynamics, coagulation abnormalities, and multiple system organ failure is caused by the release into the circulation of lipopolysaccharide (LPS), the structurally diverse component of Gram-negative bacterial outer membranes, and is responsible for 60% mortality in humans. Polymyxin B (PMB), a cyclic, cationic peptide antibiotic, neutralizes endotoxin but induces severe side effects in the process. The potent endotoxin neutralizing ability of PMB, however, offers possibilities for designing non-toxic therapeutic agents for combating endotoxicosis. Amongst the numerous approaches for combating endotoxic shock, peptide mediated neutralization of LPS seems to be the most attractive one. The precise mode of binding of PMB to LPS and the structural features involved therein have been elucidated only recently using a variety of biophysical approaches. These suggest that efficient neutralization of endotoxin by PMB is not achieved by mere binding to LPS but requires its sequestration from the membrane. Incorporation of this feature into the design of endotoxin neutralizing peptides should lead to the development of effective antidotes for endotoxic shock

Invariance and variability in bacterial PanK: a study based on the crystal structure of Mycobacterium tuberculosis PanK

Pantothenate kinase ( PanK) is a ubiquitous and essential enzyme that catalyzes the first step of the universal coenzyme A biosynthetic pathway. In this step, pantothenate ( vitamin B-5) is converted to 4'-phosphopantothenate, which subsequently forms coenzyme A in four enzymatic steps. The complex of this enzyme from Mycobacterium tuberculosis ( MtPanK) with a derivative of the feedback inhibitor coenzyme A has been crystallized in two forms and its structure solved. The structure was refined in both forms using room-temperature and low-temperature X-ray data. In both forms, the MtPanK subunit has a mononucleotide-binding fold with a seven-stranded central beta-sheet and helices on either side. However, there is a small though significant difference in subunit association between the two forms. The structure is also grossly similar to the enzyme from Escherichia coli. The active-site pocket and the dimeric interface are on two opposite sides of the PanK subunit. The enzymes from M. tuberculosis and E. coli exhibit several differences, particularly at the dimeric interface. On the other hand, the coenzyme A-binding region is almost entirely conserved. A delineation of the invariant and variable features of the PanK structure further indicates that the dimeric interface is very variable, while the coenzyme A-binding site is substantially invariant. A sequence alignment involving various bacterial PanKs is in agreement with this conclusion. The strong correlation between structural plasticity, evolutionary conservation and variability and function exhibited by the molecule could be important in the design of species-specific inhibitors of the enzyme

Spectral and kinetic characterization of 7,8-diaminopelargonic acid synthase from Mycobacterium tuberculosis

The indispensability of biotin for crucial processes like lipid biosynthesis coupled to the absence of the biotin biosynthesis pathway in humans make the enzymes of this pathway, attractive targets for development of novel drugs against numerous pathogens including M. tuberculosis. We report the spectral and kinetic characterization of the Mycobacterium tuberculosis 7,8-Diaminopelargonic acid (DAPA) synthase, the second enzyme of the biotin biosynthesis pathway. In contrast to the E. coli enzyme, no quinonoid intermediate was detected during the steady state reaction between the enzyme and S-adenosyl-L-methionine (SAM). The second order rate constant for this half of the reaction was determined to be 1.75±0.11 M−1s−1. The Km values for 7-keto-8-aminopelargonic acid (KAPA) and SAM are 2.83 μM and 308.28 μM, respectively whereas the Vmax and kcat values for the enzyme are 0.02074 μmoles/min/ml and 0.003 s−1, respectively. Our initial studies pave the way for further detailed mechanistic and kinetic characterization of the enzyme

Spectral and kinetic characterization of 7,8-diaminopelargonic acid synthase from Mycobacterium tuberculosis

The indispensability of biotin for crucial processes like lipid biosynthesis coupled to the absence of the biotin biosynthesis pathway in humans make the enzymes of this pathway, attractive targets for development of novel drugs against numerous pathogens including M. tuberculosis. We report the spectral and kinetic characterization of the Mycobacterium tuberculosis 7,8-Diamino-pelargonic acid (DAPA) synthase, the second enzyme of the biotin biosynthesis pathway. In contrast to the E. coli enzyme, no quinonoid intermediate was detected during the steady state reaction between the enzyme and S-adenosyl-L-methionine (SAM). The second order rate constant for this half of the reaction was determined to be 1.75 +/- 0.11 M-1 s(-1). The K-m values for 7-keto-8-aminopelargonic acid (KAPA) and SAM are 2.83 mu M and 308.28 mu M, respectively whereas the V-max and k(cat) values for the enzyme are 0.02074 mu moles/min/ml and 0.003 s(-1), respectively. Our initial studies pave the way for further detailed mechanistic and kinetic characterization of the enzyme

Polymyxin B: An ode to an old antidote for endotoxic shock

Endotoxic shock, a syndrome characterized by deranged hemodynamics, coagulation abnormalities, and multiple system organ failure is caused by the release into the circulation of lipopolysaccharide (LPS), the structurally diverse component of Gram-negative bacterial outer membranes, and is responsible for 60% mortality in humans. Polymyxin B (PMB), a cyclic, cationic peptide antibiotic, neutralizes endotoxin but induces severe side effects in the process. The potent endotoxin neutralizing ability of PMB, however, offers possibilities for designing non-toxic therapeutic agents for combating endotoxicosis. Amongst the numerous approaches for combating endotoxic shock, peptide mediated neutralization of LPS seems to be the most attractive one. The precise mode of binding of PMB to LPS and the structural features involved therein have been elucidated only recently using a variety of biophysical approaches. These suggest that efficient neutralization of endotoxin by PMB is not achieved by mere binding to LPS but requires its sequestration from the membrane. Incorporation of this feature into the design of endotoxin neutralizing peptides should lead to the development of effective antidotes for endotoxic shock

Broad substrate stereospecificity of the Mycobacterium tuberculosis 7-keto-8-aminopelargonic acid synthase: spectroscopic and kinetic studies

Biotin is an essential enzyme cofactor required for carboxylation and transcarboxylation reactions. The absence of the biotin biosynthesis pathway in humans suggests that it can be an attractive target for the development of novel drugs against a number of pathogens. 7-Keto-8-aminopelargonic acid (KAPA) synthase (EC 2.3.1.47), the enzyme catalyzing the first committed step in the biotin biosynthesis pathway, is believed to exhibit high substrate stereospecificity. A comparative kinetic characterization of the interaction of the Mycobacterium tuberculosis KAPA synthase with both L- and D-alanine was carried out to investigate the basis of the substrate stereospecificity exhibited by the enzyme. The formation of the external aldimine with D-alanine (k=82.63 M<SUP>−1</SUP> s<SUP>−1</SUP>) is ~5 times slower than that with L-alanine (k=399.4 M<SUP>−1</SUP> s<SUP>−1</SUP>). In addition to formation of the external aldimine, formation of substrate quinonoid was also observed upon addition of pimeloyl-CoA to the preformed D-alanine external aldimine complex. However, the formation of this intermediate was extremely slow compared with the substrate quinonoid with L-alanine and pimeloyl-CoA (k=16.9×10<SUP>4</SUP> M<SUP>−1</SUP> s<SUP>−1</SUP>). Contrary to earlier reports, these results clearly show that D-alanine is not a competitive inhibitor but a substrate for the enzyme and thereby demonstrate the broad substrate stereospecificity of the M. tuberculosis KAPA synthase. Further, D-KAPA, the product of the reaction utilizing D-alanine inhibits both KAPA synthase (K<SUB>i</SUB>=114.83 μM) as well as 7,8-diaminopelargonic acid synthase (IC<SUB>50</SUB>=43.9 μM), the next enzyme of the pathway