Endovenous laser ablation of saphenous veins - favorable clinical results confirm theoretical advantages of the 1940nm diode laser.

INTRODUCTION: Endovenous laser ablation (EVLA) is a recognized alternative to surgical treatment of varicose veins, although an optimal laser generator and its settings still remain a matter of debate. The aim of our study was to correlate clinical results with the theoretical advantage of the 1940nm diode laser characterized by high absorption of heat in a thin layer of coagulated tissue. METHODS: From 1/2010 to 12/2021 EVLA was performed in a total of 3529 consecutive patients with varicose veins and ultrasonographically documented superficial venous reflux of lower extremities. Three types of laser were used successively with the wavelengths of 1064 nm, 1470 nm and 1940 nm, respectively. All patients were prospectively enrolled in our registry. An early postoperative followup visit was scheduled including an assessment of venous closure; additional visits were performed only in case of complications. RESULTS: The success of venous closure did not differ (p=0.054) between the three laser types and was over 98%. The catheterbased method made it possible to perform multiple ablations in one procedure the trend was 1.08, 1.31 and 1.62. In 2021 the number of ablations per patient with the laser DL Tethys 1940 nm was 1.79. With this laser it was possible to reduce the total energy applied to one half (8 W, 5080 J/cm). The postoperative course of patients treated using the 1940nm laser was smoother - no other but the early followup visit was needed in 95.6% cases (p.

Risk stratification of neurological decompression sickness in divers.

INTRODUCTION: Patent foramen ovale (PFO) is a risk factor of decompression sickness (DCS). However, data on risk stratification of divers with a PFO are sparse. This study sought to evaluate the risk of neurological DCS (DCSneuro), based on the presence and grade of a right-to-left shunt (RLS). METHODS: A total of 640 divers were screened for a RLS using TCD between 1/2006 and 4/2017. RLS was graded as low, medium, or high grade with two subgroups - after a Valsalva maneuver or at rest. Divers were questioned about their DCS history. Survival analysis techniques were used to assess risk factors for unprovoked DCS. RESULTS: A RLS was found in 258 divers (40.3 %). 44 (17.1 %) divers with a RLS experienced DCSneuro compared to 5 (1.3 %) divers without a RLS (p <0.001). The proportion of DCSneuro increased from 4.6 % in the low-grade RLS subgroup to 57.1 % in the subgroup with high-grade RLS at rest. The hazard ratio for DCSneuro and RLS was11.806 (p <0.001). CONCLUSIONS: Divers with a RLS had a higher risk of DCSneuro and the risk increased with RLS grade. We suggest that TCD is an appropriate method for RLS screening and risk stratification in divers (Tab. 4, Fig. 2, Ref. 29).

Endovenous laser ablation of saphenous vein - mid-term results confirm permanent closure and possibility to treat more lesions in one procedure.

INTRODUCTION: Endovenous Laser Ablation (EVLA) is a common alternative to surgical treatment of varicose veins. The aim of our study was to demonstrate that laser occlusion is durable, that we can treat all patients in a one day setting, even with veins >10mm in diameter, and that multiple EVLAs can be done at the same time. METHODS: In the period from 1/2017 to 12/2019 EVLA was performed in a total of 1551 consecutive patients with varicose veins and ultrasonographically documented venous reflux. The mid-term results were evaluated in a group of patients operated from 1/2017 to 6/2017 (316 pts.). We compared a risk group that consisted of patients with veins >10mm in diameter (40 pts.) with a control group (the remaining 276 pts.). Patients with veins >10mm are traditionally considered as candidates for conventional surgery. RESULTS: The catheter-based method enabled us to perform more ablations in one procedure. In 2019 we performed 1.44 EVLA procedures per patient. There was only one postoperative follow-up visit, indicating an uncomplicated postoperative course, in 87.5% of patients of the risk group. In the control group 100% of patients had only one follow-up visit including ultrasound examination, showing an uncomplicated postoperative course (p.

Catheter laser ablation of superficial veins of the lower extremities in the symptomatic treatment of venous reflux comparison of the immediate results of two types of laser.

INTRODUCTION: Catheter-Based Endovenous Laser Ablation (EVLA) is a commonly used alternative to surgical treatment of varicose veins. Recently, catheterization methods have proved to be methods of choice due to the preference of patients who value minimal invasiveness. Research of EVLA currently focuses on optimization of the procedure, which includes study of the benefits of the individual types of laser generators and the wavelengths used. In this observational study we compared our early results in a non-selected population of consecutive patients treated with two different types of lasers. METHODS: In the period from February 2010 to June 2017, EVLA was performed in a total of 1747 consecutive patients (74% were female) with venous reflux. The average vein width was 8.5 mm (525 mm). Our study sought to compare a more economical 1470nm diode laser (DL) generator (Velas 2, China) - used to operate on 630 patients - with a Nd-Yag crystal generator (Fotona - Slovenia) used in 1117 patients. All operations were performed using the same methodology, in an outpatient setting, in one specialized center. All procedures were completed in local tumescent anesthesia under peroperative ultrasound control. Postoperative sonography was performed in all patients. RESULTS: The results did not show a statistically significant difference in early closure rates (98.8% for Nd-Yag versus 99.8 for DL p-ns). Early recurrence was observed in 9 patients (15 vein segments) and managed successfully with early re-intervention and closure in all cases. The causes of incomplete closure included mainly the known risk factors (anticoagulation therapy, history of varicophlebitis). There was no correlation with larger venous diameter. In 6 patients, thrombus prolapse was observed in the deep femoral vein lumen. All cases were successfully cured after a week of low-molecular-weight heparin therapy. Only one case of low-risk pulmonary embolism was reported in a patient who failed to follow the regime recommendations. CONCLUSION: This evidence did not show a significant difference in closure reliability and the amount of complications of the endovenous laser ablation of large and small saphenous vein with a 1060nm Nd-Yag crystal compared to the more economical 1470nm diode laser generator.

Intriguing cellular processing of a fluorinated amino acid during protein biosynthesis in Escherichia coli.

Bioincorporation of the methionine analogue S-(2-fluoroethyl)-l-homocysteine (l-MFE) into bacteriophage lysozyme overproduced in Escherichia coli results not only in the expected l-MFE incorporation but surprisingly substantial l-vinthionine incorporation into the labeled lysozymes. Synthetic l-vinthionine itself however is not activated by purified Escherichia coli methionyl-tRNA synthetase. The indirect preparation of vinthionine-containing proteins has the potential to be an alternate strategy to prepare vinyl thioether moieties for click chemistry applications on proteins.

[Patent foramen ovale and the risk of paradoxical embolization of venous bubbles in divers - cave for foam sclerotization of varicose veins]. / Perzistence foramen ovale patens a riziko paradoxní embolizace zilních bublin u potápecu - cave pro sklerotizaci varixu penovou metodou.

INTRODUCTION: Foam sclerotization of varicose veins may cause paradoxical embolization through patent foramen ovale (PFO). The aim of our study was to: 1) select an optimal screening method for the detection of PFO; 2) determine the prevalence of PFO in a non-selected population; and 3) test the risk of paradoxical embolization of venous bubbles in patients with PFO. MATERIALS AND METHODS: A diver after decompression is a suitable model for determining the risk of paradoxical embolization of venous gas bubbles. 329 Czech divers were screened for PFO. In a pilot study, we compared Transcranial Doppler Sonography (TCD) with Transesophageal Echocardiography (TEE) in 100 patients. TCD alone was used for further screening. In 31 divers with PFO, nitrogen bubbles were detected after simulated dives. Transthoracic Echocardiography (TTE) was used to detect venous bubbles in right-sided heart chambers; TTE and TCD were used to detect arterial bubbles. The right-to-left shunt was rated as non-significant (<20 arterial bubbles) or significant (20 arterial bubbles). Different decompression regimens were compared. RESULTS: In the pilot study, TCD was compared with the gold standard in PFO detection - TEE. The negative predictive value of TCD was 100%, positive predictive value was 92%. Screening was performed in a total of 329 divers, PFO was detected in 85 (25%), significant R-L shunt in 45 (14%). In simulated dive to 50 m maximum depth, venous nitrogen bubbles were detected in 7/8 (88%) divers. In 6/8 (75%) divers, paradoxical embolization was confirmed - nitrogen bubbles were detected in the systemic circulation. CONCLUSION: PFO prevalence with significant R-L shunt was 14% in the non-selected population of Czech divers. Simulated dives indicate that PFO represents a risk factor for paradoxical embolization of gas bubbles. TCD is a suitable screening method for the detection of PFO and the evaluation of R-L shunt significance. These results are indicative of a possible high risk of paradoxical embolization of gas bubbles and the trombogenic substance in patients with a larger PFO and significant R-L shunt undergoing foam sclerotization of varicose veins.

Trypanosomatid parasites causing neglected diseases.

Parasitic diseases such as Kala azar (visceral leishmaniasis), Chagas disease human (American trypanosomiasis) and African sleeping sickness (African trypanosomiasis) are affecting more than 27 million people worldwide. They are categorized amongst the most important neglected diseases causing approximately 150,000 deaths annually. As no vaccination is available, treatment is solely dependent on chemotherapeutic drugs. This review provides a comprehensive insight into the treatment of Kala azar, Chagas disease and African sleeping sickness. In addition to established drugs, novel small molecule- based therapeutic approaches are discussed. Drugs currently used for the treatment of Kala azar include pentavalent antimonials, Amphotericin B, Miltefosine, and Paromomycin. Liposomal formulations such as AmBisome provide promising alternatives. Furthermore, antiproliferative compounds might open new avenues in Kala azar treatment. Regarding Chagas disease, chemotherapy is based on two drugs, Nifurtimox and Benznidazole. However, sequencing of T. cruzi genome in the year 2005 raises a hope for new drug targets. Proteases, sterols and sialic acids are potential promising drug targets. Suramin, Pentamidine, Melarsporol and Eflornithine are well-established drugs to treat African sleeping sickness. New treatment options include combination therapy of Eflornithine and Nifurtimox, a Chagas disease therapeutic.. However, all approved chemotherapeutic compounds for trypanosomatid diseases suffer from high toxicity. Further, increasing resistance limits their efficacy and compliance.

Investigation of bioisosteric effects on the interaction of substrates/ inhibitors with the methionyl-tRNA synthetase from Escherichia coli.

Aminoacyl-tRNA synthetases catalyze the stepwise coupling of specific amino acid substrates to their cognate tRNAs. The first intermediate formed in this process is the aminoacyl-adenylate, which then subsequently reacts with the 3'-terminus of the cognate tRNA to transfer the amino acid to the tRNA. This overall reaction is critical for protein biosynthesis and is quintessential to the viability of all organisms. Therefore, the selective inhibition of bacterial amino acid-tRNA synthetases is the focus of intense current interest for the development of novel antibacterial agents. In order to elucidate some of the critical factors involved in recognition and binding of potential inhibitors to these bacterial systems, the current report has focused on the methionyl-tRNA synthetase from Escherichia coli. This enzyme has been studied with two sets of bioisosteric replacements in the methionine and methionyl-adenylate structures. Replacements of the carboxyl group of methionine with the phosphinic and phosphonic acid moieties were used to probe the effects of including potential transition state analogs on enzyme inhibition. The contributions of the aminoacyl-adenylate structure and the effect that fluorination has on inhibitory activity were investigated utilizing 5'-O-[(L-methionyl)-sulfamoyl]adenosine and 5'-O-[(S-trifluoromethyl-L-homocysteinyl)-sulfamoyl]adenosine. The K(i) values for these compounds were determined to be 0.4 mM, 1.2 mM, 0.25 nM and 2.4 nM respectively. A discussion of this data in relation to structural information provided by the recent determination of the three-dimensional structures of the E. coli enzyme with several of these compounds is presented.

Elucidation of solvent exposure, side-chain reactivity, and steric demands of the trifluoromethionine residue in a recombinant protein.

When incorporated into proteins, fluorinated amino acids have been utilized as 19F NMR probes of protein structure and protein-ligand interactions, and as subtle structural replacements for their parent amino acids which is not possible using the standard 20-amino acid repertoire. Recent investigations have shown the ability of various fluorinated methionines, such as difluoromethionine (DFM) and trifluoromethionine (TFM), to be bioincorporated into recombinant proteins and to be extremely useful as 19F NMR biophysical probes. Interestingly, in the case of the bacteriophage lambda lysozyme (LaL) which contains only three Met residues (at positions 1, 14, and 107), four 19F NMR resonances are observed when TFM is incorporated into LaL. To elucidate the underlying structural reasons for this anomalous observation and to more fully explore the effect of TFM on protein structure, site-directed mutagenesis was used to assign each 19F NMR resonance. Incorporation of TFM into the M14L mutant resulted in the collapse of the two 19F resonances associated with TFM at position 107 into a single resonance, suggesting that when position 14 in wild-type protein contains TFM, a subtle but different environment exists for the methionine at position 107. In addition, 19F and [1H-13C]-HMQC NMR experiments have been utilized with paramagnetic line broadening and K2PtCl4 reactivity experiments to obtain information about the probable spatial position of each Met in the protein. These results are compared with the recently determined crystal structure of LaL and allow for a more detailed structural explanation for the effect of fluorination on protein structure.

Crystal structure of the lytic transglycosylase from bacteriophage lambda in complex with hexa-N-acetylchitohexaose.

The three-dimensional structure of the lytic transglycosylase from bacteriophage lambda, also known as bacteriophage lambda lysozyme, complexed to the hexasaccharide inhibitor, hexa-N-acetylchitohexaose, has been determined by X-ray crystallography at 2.6 A resolution. The unit cell contains two molecules of the lytic transglycosylase with two hexasaccharides bound. Each enzyme molecule is found to interact with four N-acetylglucosamine units from one hexasaccharide (subsites A-D) and two N-acetylglucosamine units from the second hexasaccharide (subsites E and F), resulting in all six subsites of the active site of this enzyme being filled. This crystallographic structure, therefore, represents the first example of a lysozyme in which all subsites are occupied, and detailed protein-oligosaccharide interactions are now available for this bacteriophage lytic transglycosylase. Examination of the active site furthermore reveals that of the two residues that have been implicated in the reaction mechanism of most other c-type lysozymes (Glu35 and Asp52 in hen egg white lysozyme), only a homologous Glu residue is present. The lambda lytic transglycosylase is therefore functionally closely related to the Escherichia coli Slt70 and Slt35 lytic transglycosylases and goose egg white lysozyme which also lack the catalytic aspartic acid.

Characterization of yeast homoserine dehydrogenase, an antifungal target: the invariant histidine 309 is important for enzyme integrity.

Fungal homoserine dehydrogenase (HSD) is required for the biosynthesis of threonine, isoleucine and methionine from aspartic acid, and is a target for antifungal agents. HSD from the yeast Saccharomyces cerevisiae was overproduced in Escherichia coli and 25 mg of soluble dimeric enzyme was purified per liter of cell culture in two steps. HSD efficiently reduces aspartate semialdehyde to homoserine (Hse) using either NADH or NADPH with kcat/Km in the order of 10(6-7) M(-1) x s(-1) at pH 7.5. The rate constant of the reverse direction (Hse oxidation) was also significant at pH 9.0 (kcat/Km approximately 10(4-5) M(-1) x s(-1)) but was minimal at pH 7.5. Chemical modification of HSD with diethyl pyrocarbonate (DEPC) resulted in a loss of activity that could be obviated by the presence of substrates. UV difference spectra revealed an increase in absorbance at 240 nm for DEPC-modified HSD consistent with the modification of two histidines (His) per subunit. Amino acid sequence alignment of HSD illustrated the conservation of two His residues among HSDs. These residues, His79 and His309, were substituted to alanine (Ala) using site directed mutagenesis. HSD H79A had similar steady state kinetics to wild type, while kcat/Km for HSD H309A decreased by almost two orders of magnitude. The recent determination of the X-ray structure of HSD revealed that His309 is located at the dimer interface [B. DeLaBarre, P.R. Thompson, G.D. Wright, A.M. Berghuis, Nat. Struct. Biol. 7 (2000) 238-244]. The His309Ala mutant enzyme was found in very high molecular weight complexes rather than the expected dimer by analytical gel filtration chromatography analysis. Thus the invariant His309 plays a structural rather than catalytic role in these enzymes.

An XAS investigation of product and inhibitor complexes of Ni-containing GlxI from Escherichia coli: mechanistic implications.

Escherichia coli glyoxalase I (GlxI) is a metalloisomerase that is maximally activated by Ni(2+), unlike other known GlxI enzymes which are active with Zn(2+). The metal is coordinated by two aqua ligands, two histidines (5 and 74), and two glutamates (56 and 122). The mechanism of E. coli Ni-GlxI was investigated by analyzing Ni K-edge X-ray absorption spectroscopic (XAS) data obtained from the enzyme and complexes formed with the product, S-D-lactoylglutathione, and various inhibitors. The analysis of X-ray absorption near edge structure (XANES) was used to determine the coordination number and geometry of the Ni site in the various Ni-GlxI complexes. Metric details of the Ni site structure were obtained from the analysis of extended X-ray absorption fine structure (EXAFS). Interaction of S-D-lactoylglutathione (product) or octylglutathione with the enzyme did not change the structure of the Ni site. However, analysis of XAS data obtained from a complex formed with a peptide hydroxamate bound to Ni-GlxI is consistent with this inhibitor binding to the Ni center by displacement of both water molecules. XANES analysis of this complex is best fit with a five-coordinate metal and, given the fact that both histidine ligands are retained, suggests the loss of a glutamate ligand. The loss of a glutamate ligand would preserve the neutral charge on the Ni complex and is consistent with the lack of a significant shift in the Ni K-edge energy in this complex. These data are compared with data obtained from the E. coli Ni-GlxI selenomethionine-substituted enzyme. The replacement of three methionine residues in the native enzyme with selenomethionine does not affect the structure of the Ni site. However, addition of the peptide hydroxamate inhibitor leads to the formation of a complex whose structure as determined by XAS analysis is consistent with inhibitor binding via displacement of both water molecules but retention of both histidine and glutamate ligands. This leads to an anionic complex, which is consistent with an observed 1.7 eV decrease in the Ni K-edge energy. Plausible reaction mechanisms for Ni-GlxI are discussed in light of the structural information available.

Determination of the structure of Escherichia coli glyoxalase I suggests a structural basis for differential metal activation.

The metalloenzyme glyoxalase I (GlxI) converts the nonenzymatically produced hemimercaptal of cytotoxic methylglyoxal and glutathione to nontoxic S-D-lactoylglutathione. Human GlxI, for which the structure is known, is active in the presence of Zn(2+). Unexpectedly, the Escherichia coli enzyme is inactive in the presence of Zn(2+) and is maximally active with Ni(2+). To understand this difference in metal activation and also to obtain a representative of the bacterial enzymes, the structure of E. coli Ni(2+)-GlxI has been determined. Structures have also been determined for the apo enzyme as well as complexes with Co(2+), Cd(2+), and Zn(2+). It is found that each of the protein-metal complexes that is catalytically active has octahedral geometry. This includes the complexes of the E. coli enzyme with Ni(2+), Co(2+), and Cd(2+), as well as the structures reported for the human Zn(2+) enzyme. Conversely, the complex of the E. coli enzyme with Zn(2+) has trigonal bipyramidal coordination and is inactive. This mode of coordination includes four protein ligands plus a single water molecule. In contrast, the coordination in the active forms of the enzyme includes two water molecules bound to the metal ion, suggesting that this may be a key feature of the catalytic mechanism. A comparison of the human and E. coli enzymes suggests that there are differences between the active sites that might be exploited for therapeutic use.

Thiol-disulfide exchange is involved in the catalytic mechanism of peptide methionine sulfoxide reductase.

Peptide methionine sulfoxide reductase (MsrA; EC ) reverses the inactivation of many proteins due to the oxidation of critical methionine residues by reducing methionine sulfoxide, Met(O), to methionine. MsrA activity is independent of bound metal and cofactors but does require reducing equivalents from either DTT or a thioredoxin-regenerating system. In an effort to understand these observations, the four cysteine residues of bovine MsrA were mutated to serine in a series of permutations. An analysis of the enzymatic activity of the variants and their free sulfhydryl states by mass spectrometry revealed that thiol-disulfide exchange occurs during catalysis. In particular, the strictly conserved Cys-72 was found to be essential for activity and could form disulfide bonds, only upon incubation with substrate, with either Cys-218 or Cys-227, located at the C terminus. The significantly decreased activity of the Cys-218 and Cys-227 variants in the presence of thioredoxin suggested that these residues shuttle reducing equivalents from thioredoxin to the active site. A reaction mechanism based on the known reactivities of thiols with sulfoxides and the available data for MsrA was formulated. In this scheme, Cys-72 acts as a nucleophile and attacks the sulfur atom of the sulfoxide moiety, leading to the formation of a covalent, tetracoordinate intermediate. Collapse of the intermediate is facilitated by proton transfer and the concomitant attack of Cys-218 on Cys-72, leading to the formation of a disulfide bond. The active site is returned to the reduced state for another round of catalysis by a series of thiol-disulfide exchange reactions via Cys-227, DTT, or thioredoxin.

Identification of sequences encoding the detoxification metalloisomerase glyoxalase I in microbial genomes from several pathogenic organisms.

The ubiquitous glyoxalase system, which is composed of two enzymes, removes cellular cytotoxic methylglyoxal (MG). In an effort to identify critical residues conserved in the evolution of the first enzyme in this system, glyoxalase I (GlxI), as well as the structural implications of sequence alterations in this enzyme, a search of the National Center for Biotechnology Information (NCBI) database of unfinished genomes was undertaken. Eleven putative GlxI sequences from pathogenic organisms were identified and analyses of these sequences in relation to the known and previously identified GlxI enzymes were performed. Several of these sequences show a very high similarity to the Escherichia coli GlxI sequence, most notably the 79% identity of the sequence identified from Yersinia pestis, the causative agent of bubonic plague. In addition to the conservation of residues critical to binding the catalytic metal in all of the proposed GlxI enzymes, four regions in the Homo sapiens GlxI enzyme are absent in all of the bacterial GlxI sequences, with the exception of Pseudomonas putida. Removal of these regions may alter the active-site conformation of the bacterial enzymes in relation to that of the H. sapiens. These differences may be targeted for the development of inhibitors selective to the bacterial enzymes.

Characterization of glyoxalase I (E. coli)-inhibitor interactions by electrospray time-of-flight mass spectrometry and enzyme kinetic analysis.

Potential inhibitors of the enzyme glyoxalase I from Escherichia coli have been evaluated using a combination of electrospray mass spectrometry and conventional kinetic analysis. An 11-membered library of potential inhibitors included a glutathione analogue resembling the transition-state intermediate in the glyoxalase I catalysis, several alkyl-glutathione, and one flavonoid. The E. coli glyoxalase I quaternary structure was found to be predominantly dimeric, as is the homologous human glyoxalase I. Binding studies by electrospray revealed that inhibitors bind exclusively to the dimeric form of glyoxalase I. Two specific binding sites were observed per dimer. The transition-state analogue was found to have the highest binding affinity, followed by a newly identified inhibitor; S-(2-[3-(hexyloxy)benzoyl]-vinyl)glutathione. Kinetic analysis confirmed that the order of affinity established by mass spectrometry could be correlated to inhibitory effects on the enzymatic reaction. This study shows that selective inhibitors may exist for the E. coli homologue of the glyoxalase I enzyme.

Insights into the mechanism of Escherichia coli methionine aminopeptidase from the structural analysis of reaction products and phosphorus-based transition-state analogues.

In an effort to differentiate between alternative mechanistic schemes that have been postulated for Escherichia coli methionine aminopeptidase (eMetAP), the modes of binding of a series of products and phosphorus-based transition-state analogues were determined by X-ray crystallography. Methionine phosphonate, norleucine phosphonate, and methionine phosphinate bind with the N-terminal group interacting with Co2 and with the respective phosphorus oxygens binding between the metals, interacting in a bifurcated manner with Co1 and His178 and hydrogen bonded to His79. In contrast, the reaction product methionine and its analogue trifluoromethionine lose interactions with Co1 and His79. The interactions with the transition-state analogues are, in general, very similar to those seen previously for the complex of the enzyme with a bestatin-based inhibitor. The mode of interaction of His79 is, however, different. In the case of the bestatin-based inhibitor, His79 interacts with atoms in the peptide bond between the P(1)' and P(2)' residues. In the present transition-state analogues, however, the histidine moves 1.2 A toward the metal center and hydrogen bonds with the atom that corresponds to the nitrogen of the scissile peptide bond (i.e., between the P(1) and P(1)' residues). These observations tend to support one of the mechanistic schemes for eMetAP considered before, although with a revision in the role played by His79. The results also suggest parallels between the mechanism of action of methionine aminopeptidase and other "pita-bread" enzymes including aminopeptidase P and creatinase.

Identification of glyoxalase I sequences in Brassica oleracea and Sporobolus stapfianus: evidence for gene duplication events.

Glyoxalase I (GlxI) is the first of two enzymes involved in the cellular detoxification of methylglyoxal. A recent search of the National Center for Biotechnology Information (NCBI) databases with the protein sequence of Salmonella typhimurium GlxI identified two new hypothetical proteins with unassigned function. These two sequences, from Brassica oleracea and Sporobolus stapfianus, have significant sequence similarity to known GlxI sequences, suggesting that these two open reading frames encode for GlxI in these plants. Interestingly, analysis of these two new sequences indicates that they code for a protein composed of two fused monomers, a situation previously found solely in the yeast GlxI enzymes.

Overproduction and characterization of a dimeric non-zinc glyoxalase I from Escherichia coli: evidence for optimal activation by nickel ions.

The ubiquitous glyoxalase system converts toxic alpha-keto aldehydes into their corresponding nontoxic 2-hydroxycarboxylic acids, utilizing glutathione (GSH) as a cofactor. The first enzyme in this system, glyoxalase I (GlxI), catalyzes the isomerization of the hemithioacetal formed nonenzymatically between GSH and cytotoxic alpha-keto aldehydes. To study the Escherichia coli GlxI enzyme, the DNA encoding this protein, gloA, was isolated and incorporated into the plasmid pTTQ18. Nucleotide sequencing of the gloA gene predicted a polypeptide of 135 amino acids and Mr of 14 919. The gloA gene has been overexpressed in E. coli and shown to encode for GlxI. An effective two-step purification protocol was developed, yielding 150-200 mg of homogeneous protein per liter of culture. Electrospray mass spectrometry confirmed the monomeric weight of the purified protein, while gel filtration analysis indicated GlxI to be a homodimer of 30 kDa. Zinc, the natural metal ion found in the Homo sapiens and Saccharomyces cerevisiae GlxI, had no effect on the activity of E. coli GlxI. In contrast, the addition of NiCl2 to the growth medium or to purified E. coli apo-GlxI greatly enhanced the enzymatic activity. Inductively coupled plasma and atomic absorption analyses indicated binding of only one nickel ion per dimeric enzyme, suggesting only one functional active site in this homodimeric enzyme. In addition, the apoprotein regained maximal activity with one molar equivalence of nickel chloride, indicative of tight metal binding. The effects of pH on the kinetics of the nickel-activated enzyme were also studied. This is the first example of a non-zinc activated GlxI whose maximal activation is seen with Ni2+.