Hepatic artery ligation for arterial rupture following liver transplantation: a reasonable option.

Hepatic artery (HA) rupture after liver transplantation is a rare complication with high mortality. This study aimed to review the different managements of HA rupture and their results. From 1997 to 2007, data from six transplant centers were reviewed. Of 2649 recipients, 17 (0.64%) presented with HA rupture 29 days (2-92) after transplantation. Initial management was HA ligation in 10 patients, reanastomosis in three, aorto-hepatic grafting in two and percutaneous arterial embolization in one. One patient died before any treatment could be initiated. Concomitant biliary leak was present in seven patients and could be subsequently treated by percutaneous and/or endoscopic approaches in four patients. Early mortality was not observed in patients with HA ligation and occurred in 83% of patients receiving any other treatment. After a median follow-up of 70 months, 10 patients died (4 after retransplantation), and 7 patients were alive without retransplantation (including 6 with HA ligation). HA ligation was associated with better 3-year survival (80% vs. 14%; p=0.002). Despite its potential consequences on the biliary tract, HA ligation should be considered as a reasonable option in the initial management for HA rupture after liver transplantation. Unexpectedly, retransplantation was not always necessary after HA ligation in this series.

Mycophenolate mofetil monotherapy for severe side effects of calcineurin inhibitors following liver transplantation.

Withdrawal of calcineurin inhibitors (CNI) followed by mycophenolate mofetil (MMF) monotherapy after liver transplantation (LT) remains controversial due to the increased risk of acute rejection and graft loss. The aim of the present study, performed in a large cohort of liver-transplanted patients with severe CNI-induced side effects, was to assess renal function recovery, and safety in terms of liver function, of complete CNI withdrawal and replacement by MMF monotherapy. Fifty-two patients treated with MMF monotherapy for CNI-induced toxicity were analyzed. Mean estimated glomerular filtration rate (eGFR) increased significantly during the period of MMF monotherapy, from 37 +/- 10 to 44.7 +/- 15 mL/min/1.73 m(2) at 6 months (p = 0.001) corresponding to a benefit of +17.4% in renal function. eGFR stabilized or improved in 86.5%, 81% and 79% of cases, and chronic renal dysfunction worsened in 13.5%, 19% and 21% of cases, at 6, 12 and 24 months after CNI withdrawal, respectively. Only two patients experienced acute rejection. MMF monotherapy may be efficient at reversing/stabilizing CRD, and appears relatively safe in terms of liver graft function in long-term liver-transplanted patients. However, clinicians must bear in mind the potential risk of rejection and graft loss, and should be very cautious in the management of such 'difficult-to-treat patients'.

Fatal hemophagocytic syndrome related to human herpesvirus-6 reinfection following liver transplantation: a case report.

Human herpesvirus-6 (HHV-6) has been identified as the causal agent of exanthema subitum in early childhood (also called sixth disease or roseola), a mononucleosis-like disease in adults, and as an opportunistic pathogen in transplant recipients. In the latter setting, most infections are caused by reactivation of the latent virus in the recipient and generally have a paucisymptomatic course. Only limited data on HHV-6 infection are available for liver transplant recipients. Herein we have reported a case of fatal hemophagocytic syndrome related to HHV-6 reactivation 2 weeks after liver transplantation (LT). This case suggests that this virus may be a serious and potentially life-threatening pathogen following LT.

Molecular basis for group-specific activation of the virulence regulator PlcR by PapR heptapeptides.

The transcriptional regulator PlcR and its cognate cell-cell signalling peptide PapR form a quorum-sensing system that controls the expression of extra-cellular virulence factors in various species of the Bacillus cereus group. PlcR and PapR alleles are clustered into four groups defining four pherotypes. However, the molecular basis for group specificity remains elusive, largely because the biologically relevant PapR form is not known. Here, we show that the in vivo active form of PapR is the C-terminal heptapeptide of the precursor, and not the pentapeptide, as previously suggested. Combining genetic complementation, anisotropy assays and structural analysis we provide a detailed functional and structural explanation for the group specificity of the PlcR-PapR quorum-sensing system. We further show that the C-terminal helix of the PlcR regulatory domain, specifically the 278 residue, in conjunction with the N-terminal residues of the PapR heptapeptide determines this system specificity. Variability in the specificity-encoding regions of plcR and papR genes suggests that selection and evolution of quorum-sensing systems play a major role in adaptation and ecology of Bacilli.

Renal function outcome in pediatric liver transplant recipients.

The orthotopic liver transplantation (OLT) allows survival of children followed for severe hepatic injury, provided that the immunosuppressive treatment is prolonged. The nephrotoxicity of cyclosporine predicts the long-term outcome of the adult patients receiving a liver transplant. The aim of this study was to determine the long-term outcome of renal function in children receiving OLT. This study included 12 children, with a median for age of 7.1 yr (2-15 yr) at the time of OLT. The duration of follow-up was at least 4 yr, being 7 yr in 10 patients and more than 10 yr in seven. Renal function was evaluated with the serum level of creatinine, calculated glomerular filtration rate (cGFR), and measurement of glomerular filtration rate using chrome 51 ethylenediaminetetraacetate ((51)Cr EDTA) clearance performed at least once during follow-up. The doses and the serum concentrations (C(0)) of cyclosporine were reported at each study time. The cGFR decreased significantly 2 yr after the OLT [median (range): 106 mL/min/1.73 m(2) (71-150) at the time of OLT vs. 85 mL/min/1.73 m(2) (57-128) 2 yr after the OLT, p = 0.03], and decreased again between 7 and 10 yr after OLT [median (range): 99 mL/min/1.73 m(2) (76-125) 7 yr after OLT vs. 81 mL/min/1.73 m(2) (66-140) 10 yr after OLT, p = 0.04]. Six patients developed chronic renal failure (cGFR from 57 to 80 mL/min/1.73 m(2)) 2 yr after OLT associated with high doses of cyclosporine [median (range): 8.8 mg/kg/day (3.5-13)]. The cGFR overestimated renal function by 16% compared with the isotopic measurement of GFR (p = 0.03). Using the (51)Cr EDTA measurement, six of seven patients followed up more than 10 yr after OLT presented mild (n = 3) or moderate (n = 3) chronic renal failure. In our study, the majority of OLT recipients developed a chronic renal failure 10 yr after transplantation. Cyclosporine seems to be the most important factor responsible for the impairment of renal function. The use of the mycophenolate mofetil, a new immunosuppressive agent, allowing a reduction in the dose of cyclosporine, could minimize renal dysfunction. While awaiting the results of a prospective long-term study, close drug monitoring is advised.

A natural variant of Bacillus licheniformis alpha-amylase isolated from flour mill wastewaters sheds light on the origin of high thermostability.

AIMS: Understanding the origin of high thermostability exhibited by the alpha-amylase produced by a natural strain of Bacillus licheniformis. METHODS AND RESULTS: The MSH320 alpha-amylase gene has been cloned from a native strain of B. licheniformis isolated from flour mill wastewaters in Kashan, central Iran, and its nucleotide sequence was determined (GenBank Accession Number AF438149). Whereas previously cloned B. licheniformisalpha-amylase (BLA) genes are nearly identical, the MSH320 gene coding sequence presents only 93% identity with the reference 'wild-type' BLA gene, most of the nucleotide changes leading to silent mutations. Amino acid substitutions occurred at 19 of the 483 residues of the matured protein, distributed all along the protein sequence. Nevertheless, the natural BLA variant presents thermoinactivation kinetics similar to that of the reference BLA. Protein modelling and structural predictions at the substitution sites suggest that half of the mutations may have a significant stabilizing or destabilizing effect on the protein structure. Compensatory mutations thus occurred in the natural variant in order to maintain thermostability to the level of the reference enzyme. CONCLUSIONS: The exceptional high thermostability of BLA, although produced by a nonthermophilic organism, is not fortuitous but subject to a selective pressure still at work in natural environments. SIGNIFICANCE AND IMPACT OF THE STUDY: BLA thermal performances are not naturally maximized and can be substantially improved by protein engineering.

Selection of diabetic patients for islet transplantation. A single-center experience.

OBJECTIVE: Since the Edmonton protocol, islet transplantation (IT) offers the prospect of adequate glycemic control with no major surgical risk. In our single-center experience of IT, we studied the recruitment of eligible diabetic patients. METHODS: Between 1998 and 2002, we screened 79 diabetic patients that were divided into 2 groups according to their renal status: 41 were not receiving dialysis (ND) while 38 were receiving ongoing dialysis (D). RESULTS: In the ND group, 20 patients initiated the contact with our team, 8 patients were recruited during hospitalization for very poor glycemic imbalance, and 13 were referred by their diabetologist. 14/41 (34%) patients were ineligible for IT either because of very good glycemic balance, detectable C-peptide (C-p), kidney or liver problems, or plans for future pregnancy. 16/41 (39%) did not wish to proceed, 7 of whom were more interested by a pump. 11/41 (27%) were eligible, among which 8 are currently being assessed, 1 is on the waiting list and 2 have been transplanted. In the D group, 17/38 (45%) had a detectable C-p and received a kidney graft alone. Among the remaining 21 C-p negative diabetic patients, 3 were not eligible for kidney transplantation mainly for psychological reasons, and 4 were enlisted for kidney+pancreas transplantation. The remaining 14 C-p negative patients were kidney-transplanted. Among them, 6 were not eligible for IT, mainly for lack of motivation, slightly positive C-p stimulation tests, obesity, cancer, or increased creatininemia. The remaining 8/14 C-p negative kidney-engrafted patients were enlisted for IT. 3 had secondary failure with the pre-Edmonton immunosuppressive (IS) protocol. Five have been transplanted with the Edmonton-like IS regimen. CONCLUSION: Twenty-five per cent of the 79 patients for whom islet transplantation was considered underwent pregraft assessment and 12% (10 patients, 8 kidney-transplanted and 2 islet alone) of the 79 have been transplanted. The main eligibility criteria were undetectable Cpeptide, normal kidney function, average weight, glycemic imbalance, hypoglycemia unawareness, and glycemic brittleness.

Dimer stabilization upon activation of the transcriptional antiterminator LicT.

LicT belongs to the BglG/SacY family of transcriptional antiterminators that induce the expression of sugar metabolizing operons in Gram positive and Gram negative bacteria. These proteins contain a N-terminal RNA-binding domain and a regulatory domain called PRD which is phosphorylated on conserved histidine residues by components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Although it is now well established that phosphorylation of PRD-containing transcriptional regulators tunes their functional response, the molecular and structural basis of the regulation mechanism remain largely unknown.A constitutively active LicT variant has been obtained by introducing aspartic acid in replacement of His207 and His269, the two phosphorylatable residues of the PRD2 regulatory sub-domain. Here, the functional and structural consequences of these activating mutations have been evaluated in vitro using various techniques including surface plasmon resonance, limited proteolysis, analytical centrifugation and X-ray scattering. Comparison with the native, unphosphorylated form shows that the activating mutations enhance the RNA-binding activity and induce tertiary and quaternary structural changes. Both mutant and native LicT form dimers in solution but the native dimer exhibits a less stable and more open conformation than the activated mutant form. Examination of the recently determined crystal structure of mutant LicT regulatory domain suggests that dimer stabilization is accomplished through salt-bridge formation at the PRD2:PRD2 interface, resulting in domain motion and dimer closure propagating the stabilizing effect from the protein C-terminal end to the N-terminal effector domain. These results suggest that LicT activation arises from a conformational switch inducing long range rearrangement of the dimer interaction surface, rather than from an oligomerization switch converting an inactive monomer into an active dimer.

Structural insights into the regulation of bacterial signalling proteins containing PRDs.

PRD-containing proteins are bacterial transcriptional antiterminators and activators characterised by a duplicated phosphorylation domain involved in the regulation of catabolic operons. Recent genetic and biochemical studies have suggested how the activity of these regulators is positively or negatively controlled through the multiple phosphorylation of conserved histidyl residues. The regulation mode of these proteins has been examined in light of the recently determined first crystal structure of the phosphorylatable domain of the LicT antiterminator.

Sites of positive and negative regulation in the Bacillus subtilis antiterminators LicT and SacY.

The Bacillus subtilis homologous transcriptional antiterminators LicT and SacY control the inducible expression of genes involved in aryl beta-glucoside and sucrose utilization respectively. Their RNA-binding activity is carried by the N-terminal domain (CAT), and is regulated by two similar C-terminal domains (PRD1 and PRD2), which are the targets of phosphorylation reactions catalysed by the phosphoenolpyruvate: sugar phosphotransferase system (PTS). In the absence of the corresponding inducer, LicT is inactivated by BglP, the PTS permease (EII) specific for aryl beta-glucosides, and SacY by SacX, a negative regulator homologous to the EII specific for sucrose. LicT, but not SacY, is also subject to a positive control by the general PTS components EI and HPr, which are thought to phosphorylate LicT in the absence of carbon catabolite repression. Construction of SacY/LicT hybrids and mutational analysis enabled the location of the sites of this positive regulation at the two phosphorylatable His207 and His269 within LicT-PRD2, and suggested that the presence of negative charges at these sites is sufficient for LicT activation in vivo. The BglP-mediated inhibition process was found to essentially involve His100 of LicT-PRD1, with His159 of the same domain playing a minor role in this regulation. In vitro experiments indicated that His100 could be phosphorylated directly by the general PTS proteins, this phosphorylation being stimulated by phosphorylated BglP. We confirmed that, similarly, the corresponding conserved His99 residue in SacY is the major site of the negative control exerted by SacX on SacY activity. Thus, for both antiterminators, the EII-mediated inhibition process seems to rely primarily on the presence of a negative charge at the first conserved histidine of the PRD1.

Crystal structure of an activated form of the PTS regulation domain from the LicT transcriptional antiterminator.

The transcriptional antiterminator protein LicT regulates the expression of Bacillus subtilis operons involved in beta-glucoside metabolism. It belongs to a newly characterized family of bacterial regulators whose activity is controlled by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). LicT contains an N-terminal RNA-binding domain (56 residues), and a PTS regulation domain (PRD, 221 residues) that is phosphorylated on conserved histidines in response to substrate availability. Replacement of both His207 and His269 with a negatively charged residue (aspartic acid) led to a highly active LicT variant that no longer responds to either induction or catabolite repression signals from the PTS. In contrast to wild type, the activated mutant form of the LicT regulatory domain crystallized easily and provided the first structure of a PRD, determined at 1.55 A resolution. The structure is a homodimer, each monomer containing two analogous alpha-helical domains. The phosphorylation sites are totally buried at the dimer interface and hence inaccessible to phosphorylating partners. The structure suggests important tertiary and quaternary rearrangements upon LicT activation, which could be communicated from the protein C-terminal end up to the RNA-binding domain.

Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase.

Bacillus licheniformis alpha-amylase (BLA) is a starch-degrading enzyme that is highly thermostable although it is produced by a rather mesophilic organism. Over the last decade, the origin of BLA thermal properties has been extensively investigated in both academic and industrial laboratories, yet it is poorly understood. Here, we have used structure-based mutagenesis in order to probe the role of amino acid residues previously proposed as being important for BLA thermostability. Residues involved in salt-bridges, calcium binding or potential deamidation processes have been selected and replaced with various amino acids using a site-directed mutagenesis method, based on informational suppression. A total of 175 amylase variants were created and analysed in vitro. Active amylase variants were tested for thermostability by measuring residual activities after incubation at high temperature. Out of the 15 target residues, seven (Asp121, Asn126, Asp164, Asn192, Asp200, Asp204 and Ala269) were found to be particularly intolerant to any amino acid substitutions, some of which lead to very unstable mutant enzymes. By contrast, three asparagine residues (Asn172, Asn188 and Asn190) could be replaced with amino acid residues that significantly increase the thermostability compared to the wild-type enzyme. The highest stabilization event resulted from the substitution of phenylalanine in place of asparagine at position 190, leading to a sixfold increase of the enzyme's half-life at 80 degrees C (pH 5.6, 0.1 mM CaCl(2)). These results, combined with those of previous mutational analyses, show that the structural determinants contributing to the overall thermostability of BLA concentrate in domain B and at its interface with the central A domain. This region contains a triadic Ca-Na-Ca metal-binding site that appears extremely sensitive to any modification that may alter or reinforce the network of electrostatic interactions entrapping the metal ions. In particular, a loop spanning from residue 178 to 199, which undergoes pronounced conformational changes upon removal of calcium, appears to be the key feature for maintaining the enzyme structural integrity. Outside this region, most salt-bridges that were destroyed by mutations were found to be dispensable, except for an Asp121-Arg127 salt-bridge that contributes to the enhanced thermostability of BLA compared to other homologous bacterial alpha-amylases. Finally, our studies demonstrate that the natural resistance of BLA against high temperature is not optimized and can be enhanced further through various means, including the removal of possibly deamidating residues.

Mutation of recombinant catalytic subunit alpha of the protein kinase CK2 that affects catalytic efficiency and specificity.

In order to understand better the structural and functional relations between protein kinase CK2 catalytic subunit, the triphosphate moiety of ATP, the catalytic metal and the peptidic substrate, we built a structural model of Yarrowia lipolytica protein kinase CK2 catalytic subunit using the recently solved three-dimensional structure of the maize enzyme and the structure of cAMP-dependent protein kinase peptidic inhibitor (1CDK) as templates. The overall structure of the catalytic subunit is close to the structure solved by Niefind et al. It comprises two lobes, which move relative to each other. The peptide used as substrate is tightly bound to the enzyme, at specific locations. Molecular dynamic calculations in combination with the study of the structural model led us to identify amino acid residues close to the triphosphate moiety of ATP and a residue sufficiently far from the peptide that could be mutated so as to modify the specificity of the enzyme. Site-directed mutagenesis was used to replace by charged residues both glycine-48, a residue located within the glycine-rich loop, involved in binding of ATP phosphate moiety, and glycine-177, a residue close to the active site. Kinetic properties of purified wild-type and mutated subunits were studied with respect to ATP, MgCl(2) and protein kinase CK2 specific peptide substrates. The catalytic efficiency of the G48D mutant increased by factors of 4 for ATP and 17.5 for the RRRADDSDDDDD peptide. The mutant G48K had a low activity with ATP and no detectable activity with peptide substrates and was also inhibited by magnesium. An increased velocity of ADP release by G48D and the building of an electrostatic barrier between ATP and the peptidic substrate in G48K could explain these results. The kinetic properties of the mutant G177K with ATP were not affected, but the catalytic efficiency for the RRRADDSDDDDD substrate increased sixfold. Lysine 177 could interact with the lysine-rich cluster involved in the specificity of protein kinase CK2 towards acidic substrate, thereby increasing its activity.

RNA recognition by transcriptional antiterminators of the BglG/SacY family: functional and structural comparison of the CAT domain from SacY and LicT.

Transcriptional antiterminators of the BglG/SacY family are regulatory proteins that mediate the induction of sugar metabolizing operons in Gram-positive and Gram-negative bacteria. Upon activation, these proteins bind to specific targets in nascent mRNAs, thereby preventing abortive dissociation of the RNA polymerase from the DNA template. We have previously characterized the RNA-binding domain of SacY from Bacillus subtilis and determined its three-dimensional structure by both NMR and crystallography. In the present study, we have characterized the paralogous domain from LicT and we present the first structural comparison between two BglG/SacY family members. Similar to SacY, the RNA-binding activity of LicT is contained within the 56 N-terminal amino acid residue fragment corresponding to the so-called co-antiterminator (CAT) domain. Surface plasmon resonance affinity measurements show that, compared to SacY-CAT, LicT-CAT binds more tightly and more specifically to its cognate RNA target, with a KD value of about 10(-8) M. The crystal structure of LicT-CAT has been determined at 1.8 A resolution and compared to that of SacY-CAT. Both molecules fold as symmetrical dimers, each monomer comprising a four-stranded antiparallel beta-sheet that stacks against the beta-sheet of the other monomer in a very conserved manner. Comparison of the proposed RNA-binding surfaces shows that many of the conserved atoms concentrate in a central region across one face of the CAT dimer, whereas variable elements are mostly found at the edges. Interestingly, the electrostatic potential maps calculated for the two molecules are quite different, except for the core of the RNA-binding site, which appears essentially neutral in both structures.

Recovery after anaesthesia for pulmonary surgery: desflurane, sevoflurane and isoflurane.

We have studied maintenance and recovery profiles after general anaesthesia with sevoflurane, desflurane and isoflurane in 100 patients undergoing pulmonary surgery. End-tidal concentrations of anaesthetic required to maintain mean arterial pressure and heart rate within 20% of baseline values were 1.4 +/- 0.6% for sevoflurane, 3.4 +/- 0.9% for desflurane and 0.7 +/- 0.3% for isoflurane. The three anaesthetics had comparable haemodynamic effects and arterial oxygenation during one-lung ventilation. Emergence was twice as fast with desflurane than with sevoflurane or isoflurane (mean times to extubation: 8.9 (SD 5.0) min, 18.0 (17.0) min and 16.2 (11.0) min for desflurane, sevoflurane and isoflurane, respectively). Early recovery (Aldrete score, cognitive and psychomotor functions) was also more rapid after desflurane. In pulmonary surgery, desflurane, but not sevoflurane, allowed more rapid emergence and earlier recovery than isoflurane.

Impact of the lysine-188 and aspartic acid-189 inversion on activity of trypsin.

The impact of the charge rearrangement on the specificity of trypsin was tested by an inversion of sequence K188D/D189K maintaining the integrity of the charges of the substrate binding pocket when switching their polarity. In native trypsin, aspartate 189 situated at the bottom of the primary substrate binding pocket interacts with arginine and lysine side chains of the substrate. The kinetic parameters of the wild-type trypsin and K188D/D189K mutant were determined with synthetic tetrapeptide substrates. Compared with trypsin, the mutant K188D/D189K exhibits a 1.5- to 6-fold increase in the Km for the substrates containing arginine and lysine, respectively. This mutant shows a approximately 30-fold decrease of its k(cat) and its second-order rate constant k(cat)/Km decreases approximately 40- and 150-fold for substrates containing arginine and lysine, respectively. Hence, trypsin K188D/D189K displays a large increase in preference for arginine over lysine.

Refined solution structure and backbone dynamics of 15N-labeled C12A-p8MTCP1 studied by NMR relaxation.

MTCP1 (for Mature-T-Cell Proliferation) was the first gene unequivocally identified in the group of uncommon leukemias with a mature phenotype. The three-dimensional solution structure of the human p8MTCP1 protein encoded by the MTCP1 oncogene has been previously determined by homonuclear proton two-dimensional NMR methods at 600 MHz: it consists of an original scaffold comprising three alpha-helices, associated with a new cysteine motif. Two of the helices are covalently paired by two disulfide bridges, forming an alpha-hairpin which resembles an antiparallel coiled-coil. The third helix is orientated roughly parallel to the plane defined by the alpha-antiparallel motif and appears less well defined. In order to gain more insight into the details of this new scaffold, we uniformly labeled with nitrogen-15 a mutant of this protein (C12A-p8MTCP1) in which the unbound cysteine at position 12 has been replaced by an alanine residue, thus allowing reproducibly high yields of recombinant protein. The refined structure benefits from 211 additional NOEs, extracted from 15N-edited 3D experiments, and from a nearly complete set of phi angular restraints allowing the estimation of the helical content of the structured part of the protein. Moreover, measurements of 15N spin relaxation times and heteronuclear 15N¿1H¿NOEs provided additional insights into the dynamics of the protein backbone. The analysis of the linear correlation between J(0) and J(omega) was used to interpret relaxation parameters. It appears that the apparent relative disorder seen in helix III is not simply due to a lack of experimental constraints, but associated with substantial contributions of sub-nanosecond motions in this segment.

Activation of Bacillus licheniformis alpha-amylase through a disorder-->order transition of the substrate-binding site mediated by a calcium-sodium-calcium metal triad.

BACKGROUND: The structural basis as to how metals regulate the functional state of a protein by altering or stabilizing its conformation has been characterized in relatively few cases because the metal-free form of the protein is often partially disordered and unsuitable for crystallographic analysis. This is not the case, however, for Bacillus licheniformis alpha-amylase (BLA) for which the structure of the metal-free form is available. BLA is a hyperthermostable enzyme which is widely used in biotechnology, for example in the breakdown of starch or as a component of detergents. The determination of the structure of BLA in the metal-containing form, together with comparisons to the apo enzyme, will help us to understand the way in which metal ions can regulate enzyme activity. RESULTS: We report here the crystal structure of native, metal-containing BLA. The structure shows that the calcium-binding site which is conserved in all alpha-amylases forms part of an unprecedented linear triadic metal array, with two calcium ions flanking a central sodium ion. A region around the metal triad comprising 21 residues exhibits a conformational change involving a helix unwinding and a disorder-->order transition compared to the structure of metal-free BLA. Another calcium ion, not previously observed in alpha-amylases, is located at the interface between domains A and C. CONCLUSIONS: We present a structural description of a major conformational rearrangement mediated by metal ions. The metal induced disorder-->order transition observed in BLA leads to the formation of the extended substrate-binding site and explains on a structural level the calcium dependency of alpha-amylases. Sequence comparisons indicate that the unique Ca-Na-Ca metal triad and the additional calcium ion located between domains A and C might be found exclusively in bacterial alpha-amylases which show increased thermostability. The information presented here may help in the rational design of mutants with enhanced performance in biotechnological applications.