Admission Circulating Cell-Free DNA Levels as a Prognostic Factor in Pediatric Burns.

Background: Burn injuries in children are a major physical and psychological trauma, often a severe condition with long-term consequences. Current methods of assessing the extent of burn injuries on admission are inaccurate. Circulating cell-free DNA (cfDNA) is a potential marker of tissue damage that may be useful in burn care. Objective: To explore the use of cfDNA admission levels as a prognostic marker of pediatric burn severity and outcome. Methods: cfDNA levels of 38 pediatric burn patients (otherwise healthy) and 12 matched pediatric controls (minor elective surgery patients) admitted to our center were quantified by a direct fluorometric assay. Results: We found significantly higher admission cfDNA levels in the patient group (median 724 ng/ml, range 44-4405), compared to the control group (median 423 ng/ml, range 206-970, Mann-Whitney, P = 0.03) and a significant difference between cfDNA levels of partial-thickness burns (median 590 ng/ml, range 44-2909) and full-thickness burns (median 2394 ng/ml, range 528-4405, Mann-Whitney, P = 0.01). We also found significant correlations between cfDNA levels and hospitalization duration (Spearman, R = 0.42, P < 0.01) and undergoing surgical procedures (Spearman, R = 0.40, P < 0.01). PICU admission did not correlate to cfDNA levels (Spearman, R = 0.14, P = NS). Discussion. Admission cfDNA levels may be a valuable objective tool for assessing the severity of pediatric burn injuries on admission, including correlations with the length of hospitalization and surgical burden. Conclusion: Admission cfDNA levels may be a promising novel pediatric burn assessment method. Further investigation of cfDNA levels in healthy children standardized to age and larger cohorts are needed to establish cfDNA as a valuable prognostic factor for pediatric burn injury.

The entity of thermal-crush-avulsion hand injury (hot-press roller burns) treated with fast acting debriding enzymes (nexobrid): literature review and report of first case.

Hand burns are present in >60% of all burn cases and in fire mass casualty incidents even up to 100%. Most trauma and especially burns may be detrimental to the complex and delicate structures of the hand by direct injury, indirect BICS (Burn Induced Compartment Syndrome and interstitial high pressure) or by delayed or faulty treatment. BICS represents a special threat as the increasing swelling and oedema of the small diameter hand and forearm will exert pressure on the capillary/venous system, eventually ending in irreversible damage to the skin, nerves, muscle and vascular bed. Immediate release of constricting skin by incisional escharotomy and sometimes fasciotomy may arrest this vicious cycle: escharotomy is simple for experienced hand or burn surgeons, but they are not always present at the primary treatment site. The diagnosis of BICS is not simple either, as the direct measurement of interstitial/compartment pressure is rarely done. Burns caused by hot rollers such as industrial linen ironing machines are especially traumatic as besides the "simple" thermal burn, the hot rollers exert immense crushing pressure to the hand caught between the rollers. Over the last few years, several publications have described the role of a newly approved Bromelain derived enzymatic debriding agent (NexoBrid) for burns in general and hand burns in particular, and its ability to resolve or prevent BICS. We present a rare severe thermal/crush hand injury case where we were able to successfully treat the patient with NexoBrid enzymatic debridement-escharotomy.

Une atteinte des mains est observée dans plus de 60% des brûlures, et dans quasiment tous les cas de catastrophes. La plupart des traumaitismes, en particulier les brûlures, peuvent être délétères pour les structures complexes et délicates de la main. Ceci peut être dû à la brûlure elle même, à un syndrome compartimental (SC), à un traitement tardif ou mal conduit. Le SC est particulièrement dangereux dans les espaces ténus de l'avant bras et de la main. L'augmentation de pression consécutive à l'oedème obère la microcirculation, entraînant au bout du compte des lésions cutanées, nerveuses, vasculaires et musculaires irréversibles. La libération immédiate des tissus ainsi comprimés par incision de décharge cutanée voire aponévrotomie peut interrompre le cercle vicieux. Il s'agit d'un geste simple pour des chirurgiens spécialisés, malheureusement pas toujours disponibles sur le site du traitement initial. Le diagnostic de SC n'est pas toujours aisé si la mesure directe des pressions tissulaires n'est pas réalisée. Les brûlures dues à des rouleaux chauffés comme ceux des presses à repasser industrielles car, outre le dégagement de chaleur, les rouleaux exercent une pression intense sur la main coincée entre eux. Depuis quelques années, plusieurs articles on décrit le rôle d'un nouvel agent de débridement enzymatique à base de bromélaïne (Nexobrid) pour le traitement des brûlures dont celles des mains ainsi que ses capacité à prévenir et traiter les SC. Nous présentons ici un cas de main de presse avec utilisation, couronnée de succès, de Nexobrid.

Desmoid Tumor and Silicone Breast Implant Surgery: Is There Really a Connection? A Literature Review.

BACKGROUND: Desmoid tumors are borderline tumors of the connective tissue, arising in the musculo-aponeurotic stromal elements. A desmoid tumor (DT) has an infiltrative and locally aggressive growth pattern and usually does not metastasize; however, it has a high recurrence and complication rate. DT located in the breast (BDT) represents a rare extra-abdominal form. Recently, the presence of breast silicone implants was suggested by several researchers as a risk factor for developing BDT. OBJECTIVES: The goal of this review is to investigate the possible correlation between BDT and breast implant surgery. METHODS: We conducted a literature review of BDT-reported cases, associated with breast implant surgery. RESULTS: The search revealed 36 cases of BDT associated with silicone breast implants. CONCLUSIONS: Based on the reviewed data, the incidence of BDT following breast implant surgery is lower than BDT in the general population. At the moment, a possible association between breast implants and the development of breast desmoid tumors cannot be unequivocally confirmed. A world registry with accurate documentation of each case of BDT associated with breast implant surgery should be performed for future investigation. LEVEL OF EVIDENCE II: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Our initial learning curve in the enzymatic debridement of severely burned hands-Management and pit falls of initial treatments and our development of a post debridement wound treatment algorithm.

INTRODUCTION: Excisional surgical debridement (SD) is still the gold standard in the treatment of deeply burned hands, though the intricate anatomy is easily damaged. Previous studies demonstrated that enzymatic debridement with the bromelain debriding agent NexoBrid® (EDNX) is more selective and thus can preserve viable tissue with excellent outcome results. So far no method paper has been published presenting different treatment algorithms in this new field. Therefore our aim was to close this gap by presenting our detailed learning curve in EDNX of deeply burned hands. METHODS: We conducted a single-center prospective observational clinical trial treating 20 patients with deeply burned hands with EDNX. Different anaesthetic procedures, debridement and wound treatment algorithms were compared and main pitfalls described. RESULTS: EDNX was efficient in 90% of the treatments though correct wound bed evaluation was challenging and found unusual compared to SD. Post EDNX surprisingly the majority of the burn surface area was found overestimated (18 wounds). Finally we simplified our process and reduced treatment costs by following a modified treatment algorithm and treating under plexus anaesthesia bedside through a single nurse and one burn surgeon solely. Suprathel® could be shown to be an appropriate dressing for wound treatment after EDNX. Complete healing (less 5% rest defect) was achieved at an average of day 28. CONCLUSION: EDNX in deep burned hands is promising regarding handling and duration of the treatment, efficiency and selectivity of debridement, healing potential and early rehabilitation. Following our treatment algorithm EDNX can be performed easily and even without special knowledge in burn wound depth evaluation.

Bromelain-based enzymatic debridement and minimal invasive modality (mim) care of deeply burned hands.

The objective was to critically review the data and assess the implications of NexoBrid [NexoBrid-NXB formerly Debrase Gel Dressing-DGD]a in the special field of deep hand burns. Detailed analysis of endpoints in the treatment of hand burn patients was conducted as part of a multi-center, open label, randomized, controlled two-arm study to evaluate the safety and efficacy of NXB enzymatic debridement, comparing it to the current standard of care (SOC). These results were compared to a large cohort of patients treated with NXB in a previous, single arm study. Thirty-one burned hands were treated with NXB and 41 hand burns were in the SOC group. In the NXB group, 4 out of 31 hand burns (12.9%) required some excisional debridement compared to 29 out of the 41 (70.7%) in the SOC group (p<0.0001). Mean percentage of burn wound area excised in the NXB group was 4.4 ± 13.1% compared to 52.0 ± 41.4% in the SOC group (p<0.0001). None of the NXB-treated hands required escharotomy compared to 4 out of the 41 (9.7%) in the SOC group. NXB enzymatic debridement demonstrated a statistically significant reduction in burn wound excision and auto-grafting compared to SOC, and seems to prevent the need for emergency escharotomy. a DGD is produced by MediWound and distributed under the name NexoBrid®.

Le but était de réaliser une révision attentive des données et d'évaluer la place de Nexobrid (Nexobrid-NXB, précédemment Debrase Gel Dressing-DGD) dans l'indication particulière des brûlures profondes de la main. Une analyse détaillée des objectifs dans le traitement des brûlures de la main a été conduite en partie par une étude multicentrique, ouverte, randomisée, contrôlée avec 2 groupes pour évaluer la sécurité et l'efficacité de ce débridement enzymatique par rapport aux soins habituels (Standard of Care ou SOC). Ces résultats ont été comparés à une vaste cohorte de patients traités par NXB dans une étude précédente sur un seul groupe. 31 mains brûlées furent traitées par NXB et 41 dans le groupe SOC. Dans le groupe NXB, 4 sur 31 mains brûlées soit 12,9 % nécessitèrent une excision partielle, alors que 29 sur 41 dans le groupe SOC (70,7 %) (p < 0,0001). La moyenne des zones brûlées excisées dans le groupe NXB était de 4,4 (+ ou - 13,1 %) comparée aux 52,0 (+ ou - 41,4 % du groupe SOC) (p <0,0001). Aucune des mains traitées par NXB ont nécessité une excision totale, comparée à 4 sur 41 du groupe SOC (9,7 % du groupe). Le débridement enzymatique NXB montre une réduction statistiquement significative de l'indication d'excision avec autogreffe par rapport au groupe traité classiquement et semble prévenir la nécessité d'une escarrotomie en urgence.

A new bromelain-based enzyme for the release of Dupuytren's contracture: Dupuytren's enzymatic bromelain-based release.

OBJECTIVES: Injectable Bromelain Solution (IBS) is a modified investigational derivate of the medical grade bromelain-debriding pharmaceutical agent (NexoBrid) studied and approved for a rapid (four-hour single application), eschar-specific, deep burn debridement. We conducted an ex vivo study to determine the ability of IBS to dissolve-disrupt (enzymatic fasciotomy) Dupuytren's cords. MATERIALS AND METHODS: Specially prepared medical grade IBS was injected into fresh Dupuytren's cords excised from patients undergoing surgical fasciectomy. These cords were tested by tension-loading them to failure with the Zwick 1445 (Zwick GmbH & Co. KG, Ulm, Germany) tension testing system. RESULTS: We completed a pilot concept-validation study that proved the efficacy of IBS to induce enzymatic fasciotomy in ten cords compared with control in ten cords. We then completed a dosing study with an additional 71 cords injected with IBS in descending doses from 150 mg/cc to 0.8 mg/cc. The dosing study demonstrated that the minimal effective dose of 0.5 cc of 6.25 mg/cc to 5 mg/cc could achieve cord rupture in more than 80% of cases. CONCLUSIONS: These preliminary results indicate that IBS may be effective in enzymatic fasciotomy in Dupuytren's contracture.Cite this article: Dr G. Rubin. A new bromelain-based enzyme for the release of Dupuytren's contracture: Dupuytren's enzymatic bromelain-based release. Bone Joint Res 2016;5:175-177. DOI: 10.1302/2046-3758.55.BJR-2016-0072.

In vivo burn diagnosis by camera-phone diffuse reflectance laser speckle detection.

Burn diagnosis using laser speckle light typically employs widefield illumination of the burn region to produce two-dimensional speckle patterns from light backscattered from the entire irradiated tissue volume. Analysis of speckle contrast in these time-integrated patterns can then provide information on burn severity. Here, by contrast, we use point illumination to generate diffuse reflectance laser speckle patterns of the burn. By examining spatiotemporal fluctuations in these time-integrated patterns along the radial direction from the incident point beam, we show the ability to distinguish partial-thickness burns in a porcine model in vivo within the first 24 hours post-burn. Furthermore, our findings suggest that time-integrated diffuse reflectance laser speckle can be useful for monitoring burn healing over time post-burn. Unlike conventional diffuse reflectance laser speckle detection systems that utilize scientific or industrial-grade cameras, our system is designed with a camera-phone, demonstrating the potential for burn diagnosis with a simple imager.

Minimally invasive burn care: a review of seven clinical studies of rapid and selective debridement using a bromelain-based debriding enzyme (Nexobrid®).

Current surgical and non-surgical eschar removal-debridement techniques are invasive or ineffective. A bromelainbased rapid and selective enzymatic debriding agent was developed to overcome these disadvantages and compared with the standard of care (SOC). The safety and efficacy of a novel Debriding Gel Dressing (DGD) was determined in patients with deep partial and full thickness burns covering up to 67% total body surface area (TBSA). This review summarizes data from seven studies, four of which were randomized clinical trials that included a SOC or control vehicle. DGD eschar debridement efficacy was >90% in all studies, comparable to the SOC and significantly greater than the control vehicle. The total area excised was less in patients treated with DGD compared with the control vehicle (22.9% vs. 73.2%, P<0.001) or the surgical/non-surgical SOC (50.5%, P=0.006). The incidence of surgical debridement in patients treated with DGD was lower than the SOC (40/163 [24.5%] vs. 119/170 [70.0%], P0.001). Less autografting was used in all studies. Long-term scar quality and function were similar in DGD- and SOCtreated. DGD is a safe and effective method of burn debridement that offers an alternative to surgical and non-surgical SOC.

Les protocoles actuels de détersion d'une brûlure, chirurgicaux et non chirurgicaux, sont soit invasifs soit inefficaces. Un enzyme détersif rapide et spécifique, dérivé de la bromélaïne, a été développé dans le but de palier à ces 2 inconvénients. Il a été comparé aux techniques usuelles (TU). L'efficacité et l'innocuité d'un Gel Topique Détersif (GTD) ont été évaluées chez des patients souffrant de brûlures intermédiaires et profondes atteignant jusqu'à 67% de la Surface Corporelle Totale (SCT). Cette revue compile les données de 7 études cliniques, dont 4, randomisées, faisaient appel aux TU ou à un groupe contrôle. La détersion obtenue avec GTD était toujours > 90%, comparable aux TU et meilleure que dans le groupe contrôle. La surface relative excisée totale était moindre après GTD que chez les contrôles (22.9% VS 73.2%, p<0,001) ou les patients sous TU (50.5%, p=0,006). Le nombre de patients ayant eu besoin de chirurgie a été inférieur dans le groupe GTD que dans le groupe TU (40/163 [24.5%] VS 119/170 [70%], p<0,001). Le recours aux greffes était moins fréquent dans toutes les études. Les qualités cicatricielle et fonctionnelle à distance étaient comparables après TU et GTD. GTD est une technique de détersion efficace et sûre qui offre une alternative au TU, chirurgical ou non.

Wound 'dechronification' with negatively-charged polystyrene microspheres: a double-blind RCT.

OBJECTIVE: To compare the efficacy and safety of negatively-charged polystyrene microspheres (NCM)with controls (saline soaks) in the treatment of hard-to-heal wounds of various aetiologies. METHOD: Patients with one or more hard-to-heal wounds, defined as refractory to healing for at least 4 weeks, or those with exposed bone, tendon or ligament, were eligible for inclusion and were randomised to either NCM (PolyHeal; MediWound Ltd.) or controls, both applied twice daily for 4 weeks. Patients were monitored bi-weekly for an additional 8 weeks, while treated by standard wound care, at the investigators' discretion, and were re-evaluated 2 years after inclusion. The primary endpoint was defined as coverage of> 75% of the wound area by light-red granulation tissue after 4 weeks of treatment. RESULTS: Fifty-eight patients completed the study, 32 in the NCM group and 26 in the control group. The two most common wound types were those with primary etiologies of venous insufficiency and postoperative/post trauma. In the NCM group 47% of patients achieved > 75% light red granulation tissue after 4 weeks compared with 15% of patients in the control group (p=O.O I). The mean wound surface area in the NCM group was reduced by 39.0% after 4 weeks compared with 14.9% in the control group (p=0.02).The achievement of> 75% light red granulation tissue and reduction of mean wound surface area was also observed in the two main sub-groups (venous insufficiency and postoperative/post trauma), although it was not statistically significant, possibly due to the small sample size in each sub-group. CONCLUSION: This study demonstrates that compared to control treatment, NCM treatment of hard to-heal and chronic wounds improves formation of healthy granulation tissue and reduces wound size thus in fact 'kick-starting' the healing process and 'dechronifying' chronic wounds.

Capsular flap: new applications.

UNLABELLED: Breast augmentation with silicone implants is one of the most commonly performed procedures for women seeking improvement in their body image. Because the number of breast augmentation operations is growing, the number of subsequent reoperations is increasing. Causes for repeated operative procedures include infection, capsular contracture, silicone implant replacement, and breast reaugmentation. As the average volume of silicone implants used is steadily growing according to patients' wishes and fashion requirements, the plastic surgeon often is asked to replace the silicone implants with larger implants. Replacing breast implants with a similar sized implant is a relatively simple operation, but insertion of larger implants may present a challenge due to insufficient soft tissue coverage of the lower pole of the larger breast implant, especially in thin subjects. Total coverage of the breast implant can be achieved by use of tissue substitutes (TS), such as acellular dermal matrices. Usage of TS, however, is prone to complications and very costly, and these factors influence the implementation of TS in a private practice setup. This report describes a capsular flap used to cover the lower pole of breast implants. The flap guarantees multilayered stable wound closure and prevents displacement of the inframammary fold. The capsular flap also was used to correct an inferiorly displaced inframammary fold as a consequence of the breast augmentation. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Utilization of Inferiorly Based Dermofat Flap in Breast Reconstruction after Simple Mastectomy due to Gigantomastia.

Gigantomastia (GM) is a rare disabling condition characterized by excessive breast tissue growth. To date, there is no universal classification and definition of GM. At present, GM is determined as weight over 1.5 kg per breast (Dancey et al., 2008) or 3% or more of the patient's total body weight (Dafydd et al., 2011). The lack of generally acknowledged approach regarding GM is expressed by the different methods of its treatment ranging from hormonal prescription to mastectomy and subsequent complex breast reconstruction (Shoma et al., 2011). We describe a treatment approach, including simple mastectomy and immediate breast reconstruction by an inferiorly based dermofat flap with silicone implants and nipple grafting.

Burn treatment framework in Israel.

The treatment and hospitalization policies in various hospitals in Israel are influenced by injury severity and by the existence or non-existence of a designated burn treatment body. Severely injured burn victims requiring designated burn treatment are referred to one of Israel's five major burn units located in the highest level trauma centres that have an advanced burn treatment infrastructure. This national distribution of burn centres ensures designated treatment availability in various areas according to Israeli demographics, geography, and security threats. Israel does not have an obligatory burn report policy. Implementation of a national burn repository such as that in the USA will be able to give burn treatment specialists in our country a basis for comparison of treatment standards and allow for better care for burn victims. The Israeli Burn Association has a major role in the processes discussed in the manuscript.

Structure-specificity relationships of an intracellular xylanase from Geobacillus stearothermophilus.

Geobacillus stearothermophilus T-6 is a thermophilic Gram-positive bacterium that produces two selective family 10 xylanases which both take part in the complete degradation and utilization of the xylan polymer. The two xylanases exhibit significantly different substrate specificities. While the extracellular xylanase (XT6; MW 43.8 kDa) hydrolyzes the long and branched native xylan polymer, the intracellular xylanase (IXT6; MW 38.6 kDa) preferentially hydrolyzes only short xylo-oligosaccharides. In this study, the detailed three-dimensional structure of IXT6 is reported, as determined by X-ray crystallography. It was initially solved by molecular replacement and then refined at 1.45 A resolution to a final R factor of 15.0% and an R(free) of 19.0%. As expected, the structure forms the classical (alpha/beta)(8) fold, in which the two catalytic residues (Glu134 and Glu241) are located on the inner surface of the central cavity. The structure of IXT6 was compared with the highly homologous extracellular xylanase XT6, revealing a number of structural differences between the active sites of the two enzymes. In particular, structural differences derived from the unique subdomain in the carboxy-terminal region of XT6, which is completely absent in IXT6. These structural modifications may account for the significant differences in the substrate specificities of these otherwise very similar enzymes.

Binding of inhibitory aromatic amino acids to Streptomyces griseus aminopeptidase.

The bacterial aminopeptidase isolated from the extracellular extract of Streptomyces griseus (SGAP) is a double-zinc exopeptidase with a high preference for large hydrophobic amino-terminus residues. It is a monomer of a relatively low molecular weight (30 kDa), is heat-stable, displays a high and efficient catalytic turnover and its activity is modulated by calcium ions. Several free amino acids were found to inhibit the activity of SGAP in the millimolar concentration range and can therefore serve for the study of binding of both inhibitors and reaction products. The current study is focused on the X-ray crystallographic analysis of the SGAP complexes with L-tryptophan and p-iodo-L-phenylalanine, both at 1.30 A resolution. These two bulky inhibitory amino acids were found to bind to the active site of SGAP in very similar positions and orientations. Both of them bind to the two active-site zinc ions via their free carboxylate group, while displacing the zinc-bound water/hydroxide that is present in the native enzyme. Further stabilization of the binding of the amino-acid carboxylate group is achieved by its relatively strong interactions with the hydroxyl group of Tyr246 and the carboxylate group of Glu131. The binding is also stabilized by three specific hydrogen bonds between the amine group of the bound amino acid and enzyme residues Glu131, Asp160 and Arg202. These consistent interactions confirm the key role of these residues in the specific binding of the free amine of substrates and products, as proposed previously. The phenyl ring of Phe219 of the enzyme is involved in stacking interactions with the corresponding aromatic ring of the bound affector. This interaction seems to be important for the binding and orientation of the aromatic side chain within the specificity pocket. These structural results correlate well with the results obtained for the complexes of SGAP with other inhibitory amino acids and support the general catalytic mechanism proposed for this and related enzymes.

Structure determination of the extracellular xylanase from Geobacillus stearothermophilus by selenomethionyl MAD phasing.

Xylanases are hemicellulases that hydrolyze the internal beta-1,4-glycoside bonds of xylan. The extracellular thermostable endo-1,4-beta-xylanase (EC 3.2.1.8; XT6) produced by the thermophilic bacterium Geobacillus stearothermophilus T-6 was shown to bleach pulp optimally at pH 9 and 338 K and was successfully used in a large-scale biobleaching mill trial. The xylanase gene was cloned and sequenced. The mature enzyme consists of 379 amino acids, with a calculated molecular weight of 43 808 Da and a pI of 9.0. Crystallographic studies of XT6 were performed in order to study the mechanism of catalysis and to provide a structural basis for the rational introduction of enhanced thermostability by site-specific mutagenesis. XT6 was crystallized in the primitive trigonal space group P3(2)21, with unit-cell parameters a = b = 112.9, c = 122.7 A. A full diffraction data set for wild-type XT6 has been measured to 2.4 A resolution on flash-frozen crystals using synchrotron radiation. A fully exchanged selenomethionyl XT6 derivative (containing eight Se atoms per XT6 molecule) was also prepared and crystallized in an isomorphous crystal form, providing full selenium MAD data at three wavelengths and enabling phase solution and structure determination. The structure of wild-type XT6 was refined at 2.4 A resolution to a final R factor of 15.6% and an R(free) of 18.6%. The structure demonstrates that XT6 is made up of an eightfold TIM-barrel containing a deep active-site groove, consistent with its 'endo' mode of action. The two essential catalytic carboxylic residues (Glu159 and Glu265) are located at the active site within 5.5 A of each other, as expected for 'retaining' glycoside hydrolases. A unique subdomain was identified in the carboxy-terminal part of the enzyme and was suggested to have a role in xylan binding. The three-dimensional structure of XT6 is of great interest since it provides a favourable starting point for the rational improvement of its already high thermal and pH stabilities, which are required for a number of biotechnological and industrial applications.

Crystal structures of KDOP synthase in its binary complexes with the substrate phosphoenolpyruvate and with a mechanism-based inhibitor.

The crystal structures of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDOPS) from Escherichia coli complexed with the substrate phosphoenolpyruvate (PEP) and with a mechanism-based inhibitor (K(d) = 0.4 microM) were determined by molecular replacement using X-ray diffraction data to 2.8 and 2.3 A resolution, respectively. Both the KDOPS.PEP and KDOPS.inhibitor complexes crystallize in the cubic space group I23 with cell constants a = b = c = 117.9 and 117.6 A, respectively, and one subunit per asymmetric unit. The two structures are nearly identical, and superposition of their Calpha atoms indicates an rms difference of 0.41 A. The PEP in the KDOPS.PEP complex is anchored to the enzyme in a conformation that blocks its si face and leaves its re face largely devoid of contacts. This results from KDOPS's selective choice of a PEP conformer in which the phosphate group of PEP is extended toward the si face. Furthermore, the structure reveals that the bridging (P-O-C) oxygen atom and the carboxylate group of PEP are not strongly hydrogen-bonded to the enzyme. The resulting high degree of negative charge on the carboxylate group of PEP would then suggest that the condensation step between PEP and D-arabinose-5-phosphate (A5P) should proceed in a stepwise fashion through the intermediacy of a transient oxocarbenium ion at C2 of PEP. The molecular structural results are discussed in light of the chemically similar but mechanistically distinct reaction that is catalyzed by the enzyme 3-deoxy-D-arabino-2-heptulosonate-7-phosphate synthase and in light of the preferred enzyme-bound states of the substrate A5P.

Biochemical characterization and identification of catalytic residues in alpha-glucuronidase from Bacillus stearothermophilus T-6.

Alpha-D-glucuronidases cleave the alpha-1,2-glycosidic bond of the 4-O-methyl-D-glucuronic acid side chain of xylan, as a part of an array of xylan hydrolyzing enzymes. The alpha-D-glucuronidase from Bacillus stearothermophilus T-6 was overexpressed in Escherichia coli using the T7 polymerase expression system. The purification procedure included two steps, heat treatment and gel filtration chromatography, and provided over 0.3 g of pure enzyme from 1 L of overnight culture. Based on gel filtration, the native protein is comprised of two identical subunits. Kinetic constants with aldotetraouronic acid as a substrate, at 55 degrees C, were a Km of 0.2 mM, and a specific activity of 42 U x mg(-1) (kcat = 54.9 s(-1)). The enzyme was most active at 65 degrees C, pH 5.5-6.0, in a 10-min assay, and retained 100% of its activity following incubation at 70 degrees C for 20 min. Based on differential scanning calorimetry, the protein denatured at 73.4 degrees C. Truncated forms of the enzyme, lacking either 126 amino acids from its N-terminus or 81 amino acids from its C-terminus, exhibited low residual activity, indicating that the catalytic site is located in the central region of the protein. To identify the potential catalytic residues, site-directed mutagenesis was applied on highly conserved acidic amino acids in the central region. The replacements Glu392-->Cys and Asp364-->Ala resulted in a decrease in activity of about five orders of magnitude, suggesting that these residues are the catalytic pair.

Stereochemistry of family 52 glycosyl hydrolases: a beta-xylosidase from Bacillus stearothermophilus T-6 is a retaining enzyme.

A beta-xylosidase from Bacillus stearothermophilus T-6 assigned to the uncharacterized glycosyl hydrolase family 52 was cloned, overexpressed in Escherichia coli and purified. The enzyme showed maximum activity at 65 degrees C and pH 5.6-6.3. The stereochemistry of the hydrolysis of p-nitrophenyl beta-D-xylopyranoside was followed by 1H-nuclear magnetic resonance. Time dependent spectrum analysis showed that the configuration of the anomeric carbon was retained, indicating that a retaining mechanism prevails in family 52 glycosyl hydrolases. Sequence alignment and site-directed mutagenesis enabled the identification of functionally important amino acid residues of which Glu337 and Glu413 are likely to be the two key catalytic residues involved in enzyme catalysis.

Glutamic acid 160 is the acid-base catalyst of beta-xylosidase from Bacillus stearothermophilus T-6: a family 39 glycoside hydrolase.

A beta-xylosidase from Bacillus stearothermophilus T-6 was cloned, overexpressed in Escherichia coli and purified to homogeneity. Based on sequence alignment, the enzyme belongs to family 39 glycoside hydrolases, which itself forms part of the wider GH-A clan. The conserved Glu160 was proposed as the acid-base catalyst. An E160A mutant was constructed and subjected to steady state and pre-steady state kinetic analysis together with azide rescue and pH activity profiles. The observed results support the assignment of Glu160 as the acid-base catalytic residue.