β-Glucanases and non-cellulosic glucans in the developing oat plant

Summary The changes in the levels of various -glucan hydrolase activities in the second internode of the stem of the developing oat plant have been examined. Concurrent changes in the non-cellulosic -glucans contained in the corresponding total hemicelluloses were also studied. Possible relationships between the observed changes and the growth and development of the plant tissue are discussed.     

β-Glucanases in developing cotton (Gossypium hirsutum L.) fibres

Cotton fibres possess several -glucanase activities which appear to be associated with the cell wall, but which can be partially solubilised in buffers. The main activity detected was that of an exo-(13)--d-glucanase (EC 3.2.1.58) but which also had the characteristics of a -glucosidase (EC 3.2.1.21). Endo-(13)--d-glucanase activity (EC 3.2.1.39) and much lower levels of (14)--d-glucanase activity were also detected. The exo-(13)--glucanase showed a maximum late on (40 days post-anthesis) in the development of the fibres, whereas the endo-(13)--glucanase activity remained constant throughout fibre development. The -glucanase complex associated with the cotton-fibre cell wall also functions as a transglucosylase introducing, inter alia, (16)--glucosyl linkages into the disaccharide cellobiose to give the trisaccharide 4-O--gentiobiosylglucose.Abbreviations CMC carboxymethylcellulose - ONPG o-nitrophenyl--d-glucopyranoside - TLC thin-layer chromatography Presented at the Third Cell Wall Meeting held in Fribourg in 1984     

1,3-β-Glucanases fromStreptomyces sp.1228 lytic enzyme system

Summary Four 1,3--glucanases GI, GII, GIV and GVIII from a culture filtrate ofStreptomyces sp. 1228 were purified by anion exchange chromatography using DEAE-Sepharose Cl-6B or DEAE-Cellulose, gel filtration on Bio-Gel P-200 or Sephacryl S-200, Amicon ultrafiltration and preparative PAGE. The M_r of these enzymes were 19000, 74000, 78000 and 56000 respectively. The glucanase GVIII consisted of two subunits. The optimal catalytic activity of the purified preparations was at 50–55°C and pH 5.5–6.0. The enzymes were also most stable at this pH. Both glucanases GI and GVIII were characterized by high thermostability. The glucanases showed different affinities towards laminarin with Km values of 6.65 x 10^–5 mol/l for GI, 2.35 x 10^–4 mol/l for GII, 8.1 x 10^–5mol/l for GIV and 8.1 x 10^–4mol/l for GVIII. The presence of metal ions was not required for activity of these enzymes but thiol groups increased their activity. D-glucono--lactone did not inhibit the enzymes.     

β-1,3-Glucanases: Plasmodesmal Gate Keepers for Intercellular Communication

Plasmodesmata (Pd), coaxial membranous channels that connect adjacent plant cells, are not static, but show a dynamic nature and can be opened or closed. These controlled changes in Pd conductivity regulate plant symplasmic permeability and play a role both in development and defense processes. One of the mechanisms shown to produce these changes is the deposition and hydrolysis of callose by β-1-3-synthase and glucanase, respectively. Recently we have identified the first β-1,3-glucanase Arabidopsis enzyme that is associated to the macromolecular Pd complex, termed AtBG_pap. When fused to GFP, this previously identified GPI-anchored protein localizes to the ER and the plasma membrane where it appears in a punctuate pattern that colocalizes with callose present around Pd. In T-DNA insertion mutants that do not transcribe AtBG_pap, GFP cell-to-cell movement between epidermal cells is reduced and callose levels around Pd are elevated. In this addenda we review the plant developmental processes of symplasmic regulation that have been shown to include callose deposition and β-1,3-glucanase activity, and suggest a role for AtBG_pap in these processes. Additionally, based on the ability of viral movement proteins (MPs) to interact with ankyrin repeat proteins, and together with our recent findings showing the involvement of viral particles in callose degradation, we also purpose a new model for the ability of viruses to overcome Pd-callose deposition, and mediate their cell-to-cell movement.Key Words: plasmodesmata, cell-cell communication, callose, β-1,3-glucanase, movement protein, ankyrin repeats     

Co-Localization of β-1,3-Glucanases and Callose During Somatic Embryogenesis in Cichorium

During direct somatic embryogenesis in leaves of Cichorium hybrid clone ‘474’, 38 kDa β-1,3-glucanases are accumulated in the culture medium of the embryogenic hybrid to a higher level when compared with a non-embryogenic cultivar. In the same time, embryogenic cells were surrounded by a cell wall that was characterized by the presence of callose. This callosic deposition disappeared as embryos grew. Callose consisted of β-1,3-glucan linkages and so represented a possible substrate for β-1,3-glucanases. Using immunolocalization experiments, we demonstrated that from the three types of callose deposits observed during the culturing of Cichorium leaf explants, only the callose present in the walls surrounding reactivated cells seemed specifically related to somatic embryogenesis. Moreover, callose and the 38-kDa β-1,3-glucanases were co-localized dispersed throughout the thick and swelled walls of reactivated cells and embryo cell walls. This suggests that callose and β-1,3-glucanases are implicated in the process of somatic embryogenesis since they were always detected in or quite near embryogenic and embryo cell. This also suggested that β-1,3-glucanases could be involved in the degradation of this callose.Key Words: β-1,3-glucanases, callose, Cichorium, immunolocalizations, somatic embryogenesis     

Biochemical and Immunological Relationships Between Endo-β-1,4-Glucanases from Cockroaches

The cellulase from Geoscapheus dilatatus consisted of two major and four minor endo-β-1,4-glucanase components. Two major and one minor component were purified to homogeneity. The major endo-β-1,4-glucanase components, named GD1 and GD2, were similar to EG1 and EG2 from Panesthia cribrata in terms of M_r and kinetic properties. The purified minor component, named GD3, was distinct from GD1 and GD2 because of a lower M_r and a lower specific activity. Polyclonal antibodies raised against the two major endo-β-1,4-glucanase components, EG1 and EG2, of the cellulase from P. cribrata cross-reacted with each other and with pure GD1 and GD2 from the foregut and midgut of the related cockroach G. dilatatus but did not cross-react with GD3. Endo-β-1,4-glucanase components were partially purified from the foregut and midgut of four other cockroaches. These comprised three other Australian cockroaches also from the superfamily Blaberoidea and one American cockroach, Cryptocercus punctulatus, from the superfamily Blattoidea. The endogenous cellulases from all cockroaches examined consisted of either two or three major endo-β-1,4-glucanase components. The amino acid sequence of the N-terminus region of the two major endo-β-1,4-glucanase components from P. cribrata were determined and are homologous with those belonging to glycosyl hydrolase family 9 (cellulase family E).     

Expression of a Soluble Functional Form of the Integrin α4β1 in Mammalian Cells

The integrin α₄β₁(VLA4) has been expressed as a soluble, active, heterodimeric immunoglobulin fusion protein. cDNAs encoding the extracellular domains of the human α₄ and β₁ subunits were fused to the genomic DNA encoding the human γ1 immunoglobulin Fc domain and functional integrin fusion protein was expressed as a secreted, soluble molecule from a range of mammalian cell lines. Specific mutations were introduced into the Fc region of the molecules to promote α₄β₁ heterodimer formation. The soluble α₄β₁ Fc fusion protein exhibited divalent cation dependent binding to VCAM-1, which was blocked by the appropriate function blocking antibodies. The apparent K_d for VCAM-1 binding were similar for both the soluble and native forms of α₄β₁. In addition, the integrin–Fc fusion was shown to stain cells expressing VCAM-1 on their surface by FACs analysis. This approach for expressing soluble α₄β₁ should be generally applicable to a range of integrins.     

Role of Wall-bound β-Glucanases in Regulating Tip-growth of Lilium longiflorum Pollen Tubes

β-Glucanases were found in the cell wall of Lilium longiflorum Thunb. pollen tubes grown in vitro . The activity of β-glucanases was, in a certain extent, decreased by nojirimycin, an inhibitor of glucosidase. Pollen germination percentage reduced dramatically when nojirimycin was applied in the culture medium. In case that nojirimycin was added at 0 or 1 h after the onset of incubation, the inhibition rate was 99.6% and 91.4%, respectively. When 3 mmol/L of nojirimycin was applied in the liquid medium at 0, 1, 1.5 and 2 h after the onset of incubation, the growth of pollen tubes was interrupted, which resulted in the morphological change of the pollen tubes such as the newly grown portion of pollen tubes being bent, curved and swollen. Tracing the growth pattern of the individual pollen tube grown in semi-solid medium by video microscopy, the authors demonstrated that pollen tube growth rate was strongly inhibited by nojirimycin at concentrations ranged from 0.003 to 3 mmol/L. Moreover, the cytoplasmic arrangement and the morphology of the pollen tubes were also affected by nojirimycin. The growth inhibition brought about by nojirimycin was reversible. These results indicated that β-glucanases, which degrade 1,3-β-glucan and/or 1,4-β-glucan or 1,3:1,4-β-glucan constructed in the cell wall, are involved in pollen germination and pollen tube growth. It provides new insight into an understanding of the contribution of β-glucanases to the cell wall extensibility and the crucial role of cell wall in regards to the regulation of pollen tube growth.     

α-Amylase,Limit Dextrinase,and α-Glucosidase Enzymes in Barley and Malt

Abstract

A parallel discussion is presented of recent developments in fermentation monitoring and control in both research institutes and in industry. The discussion is based around the areas of measurement (on-line and off-line), data conditioning and analysis, modeling, fault analysis, and control. Recent progress in industrial fermentation monitoring and control is used as a guide to identify potential areas of research that might have a most rapid and direct impact on industrial fermentation operation.     

Two Pine Endo-β-1,4-Glucanases Are Associated with Rapidly Growing Reproductive Structures

Two cDNA clones encoding endo-β-1,4-glucanases (EGases) were isolated from a radiata pine (Pinus radiata) cDNA library prepared from immature female strobili. The cDNAs PrCel1 (Pinus radiata cellulase 1) and PrCel2 encode proteins 509 and 515 amino acids in length, respectively, including putative signal peptides. Both proteins contain domains conserved in plant and bacterial EGases. The proteins PRCEL1 and PRCEL2 showed strong similarity to each other (76% amino acid identity), and higher similarity to TPP18 (73 and 67%, respectively), an EGase cloned from tomato (Lycopersicon esculentum) pistils, than to any other reported EGases. Northern-blot analyses indicated that both genes displayed a similar pattern of expression. The only significant difference was in the level of expression. In situ hybridizations were used to demonstrate that, within differentiating pine reproductive structures, PrCel1 expression was greatest in microsporangia in pollen strobili and near the developing ovule in the seed strobili. Expression was also found in vegetative tissues, especially in regions experiencing cell elongation, such as the elongating region of root tips. Both proteins have an ability to degrade carboxymethylcellulose in vitro. Genomic-blot analysis indicated the presence of a family of EGase genes in the radiata pine genome, and that PrCel1 and PrCel2 are transcribed from distinct one-copy genes.The growth and development of floral organs involves many physiological processes, including modifications to the cell wall. EGases (cellulases) may play roles in cell wall loosening, which is required for expansion or major cell wall disruption. Cell expansion has been reviewed by Cosgrove (1993), who demonstrated that acid-induced extension of cell walls appears to require the activity of expansins. Wall-modifying enzymes such as endoxyloglucan transferase, and wall-degrading enzymes such as glucanases, are also likely to be involved, but there is no evidence that they can cause extension of isolated walls. Major cell wall disruption also occurs at several steps in the development of flower reproductive organs (del Campillo and Lewis, 1992). The callose wall that protects the meiotic cells is broken down during early pollen differentiation, releasing the microspores into the anther locule. Later, the tapetum begins to break down and the cytoplasm is released into the locule. Finally, the release of the mature pollen grains from the anthers is facilitated by the formation of a fissure, the stomium. Similarly, during pollen-stigma interactions, cell wall loosening of the papillary cells at the surface of the stigma has been reported. EGases have been shown to accumulate in anthers of beans and sweet peas in a developmentally regulated manner and may be involved in the cell wall disruption required for pollen differentiation.Plant EGases typically lack the ability to degrade microcrystalline cellulose in vitro. Bacterial EGases, however, are able to degrade cellulose. Therefore, all EGases are sometimes referred to as cellulases. Genes encoding EGases have been isolated from many different plant species, including tomato (Lycopersicon esculentum) (Lashbrook et al., 1994; Milligan and Gasser, 1995), elder (Sambucus nigra) (Taylor et al., 1994), pea (Pisum sativum) (Wu et al., 1996), soybean (Glycine max) (Kemmerer and Tucker, 1994), Arabidopsis (Ferl, 1995), poplar (Populus alba) (Nakamura et al., 1995), kidney bean (Phaeseolus vulgaris) (Tucker and Milligan, 1991), and avocado (Persea americana) (Tucker et al., 1987). Some of these enzymes, including TomCel2 (Lashbrook et al., 1994), EGL1 (Wu et al., 1996), and AvoCel1 (Tucker et al., 1987), are primarily associated with fruit ripening. Another group, including BAC (Tucker and Milligan, 1991), SAC1 (Kemmerer and Tucker, 1994), TomCel1 (Lashbrook et al., 1994), and JET1 (Taylor et al., 1994), are associated with abscission. Yet another group of enzymes appears to be expressed predominantly in rapidly expanding tissues. Expression of TPP18 (Milligan and Gasser, 1995), which is identical to Cel4 (Brummell et al., 1997), occurs in growing pistils of tomato flowers, and to a lesser extent in stamens, but not in fully expanded flower parts. Expression is also high in the growing zones of etiolated hypocotyls and in expanding leaves. Here we report on the expression of two EGases cloned from reproductive structures of radiata (Monterey) pine (Pinus radiata).As in angiosperms, the “flowering” of radiata pine starts with the transition of an undetermined axillary apex into a determinant reproductive apex, which develops into the strobili (cones). Reproductive buds are simple because they normally contain a single strobilus and no leaves. Mature male (pollen) cones are small (1–2 cm in length) and are made up of spirally arranged microsporophylls, each bearing two microsporangia (pollen sacs). The microspores develop into four-celled pollen grains. Female (seed) cones consist of an axis, which bears a specially arranged series of small appendages termed bracts. In the axil of each bract is a thick scale upon which two ovules are borne, attached to the adaxial surface of the cone scale near the base. Because the ovuliferous scales are lateral structures subtended by a bract, the entire cone is a “compound” strobilus, and may be compared in this respect with an inflorescence. Such female axes generally are located at the top of the adult tree, whereas male cones are located farther down the stem and contain only microsporophylls.A few genes have previously been cloned from various parts or stages of developing radiata pine cones, including the cDNAs encoding genes preferentially expressed in immature female and male cone buds. Homologs of the angiosperm late-flowering, meristem-identity, and organ-identity genes regulate development of unisexual cones in the conifer radiata pine (Mouradov et al., 1996, 1997a, 1997b). MADS box genes have also been cloned from another conifer, Norway spruce (Picea abies) (Tandre et al., 1995). Two different cDNAs with homology to legumins have been isolated from fertilized ovules of white pine (Pinus strobus), but are not expressed in unfertilized ovules (Baker et al., 1996). Several cDNA clones encoding seed-storage proteins have also been isolated from Douglas fir and interior spruce megagametophytes (Newton et al., 1992; Leal and Misra, 1993). Mature megagametophytes have been used for many years to study isozyme variation, and are commonly used as sources of DNA for genome mapping because of their haploid condition.To gain a greater understanding of the genes involved in the formation of pine reproductive structures, we constructed a cDNA library from immature female cones and differentially screened against vegetative buds. Here we report on the cloning, sequencing, and characterization of two of those genes, PrCel1 and PrCel2, that have very high homology to each other and to previously cloned EGases.     

Does Campylobacter jejuni Form Biofilms in Food-Related Environments?

Campylobacter jejuni is one of the most frequent causes of bacterial gastrointestinal food-borne infection worldwide. This species is part of the normal flora of the gastrointestinal tracts of animals used for food production, including poultry, which is regarded as the primary source of human Campylobacter infections. The survival and persistence of C. jejuni in food processing environments, especially in poultry processing plants, represent significant risk factors that contribute to the spread of this pathogen through the food chain. Compared to other food-borne pathogens, C. jejuni is more fastidious in its growth requirements and is very susceptible to various environmental stressors. Biofilm formation is suggested to play a significant role in the survival of C. jejuni in the food production and processing environment. The aims of this minireview were (i) to examine the evidence that C. jejuni forms biofilms and (ii) to establish the extent to which reported and largely laboratory-based studies of C. jejuni biofilms provide evidence for biofilm formation by this pathogen in food processing environments. Overall existing studies do not provide strong evidence for biofilm formation (as usually defined) by most C. jejuni strains in food-related environments under the combined conditions of atmosphere, temperature, and shear that they are likely to encounter. Simple attachment to and survival on surfaces and in existing biofilms of other species are far more likely to contribute to C. jejuni survival in food-related environments based on our current understanding of this species.     

Über die Form der Mitochondrien in elektronenmikroskopischen Bildern

Ohne Zusammenfassung     

Apoplastic β-1, 3-Glucanases in Leaves of Tomato Systematically Infected by TMV

Apoplastic β-1, 3-glucanase was purified from leaves of tomato (Lycopersicon esculentum Mill. ) which were systematically infected by TMV (tobacco mosaic virus). The enzyme obtained through -20℃ acetone precipitation, CM-Sephadex C-25 ion exchange chromatography, DEAE-Sephadex A-25 ion exchange chromatography and Sephadex G-75 gel filtration, showed homogeneity in PAGE, and SDS-PAGE which had two isoenzymes of 27 kD and 36 kD. The enzyme hydrolysed laminarin at an optimum pH of 4.8--5.2 and was stable between pH 4--8 and at an optimum temperature between 30--40℃, and stable at 40℃ after 1 hour of incubation, It had a Km of 9. 2 mg/mL. SDS-PAGE profiles of the proteins in the tomato leaf intercellular fluid had the bands of 22 kD, 27 kD and 36 kD that were β-1, 3-glucanases.     

Microcalorimetric Determination of the Thermostability of Three Hybrid (1–3,1–4)-β-Glucanases

Abstract

Thermodynamic parameters of the three hybrid (1–3,1–4)-β-glucanases H(A12-M), H(A12-M)ΔY13, and H(A16-M) composed of short N-terminal regions derived from the Bacillus amyloliquefaciens enzyme and a C-terminal region of the homologous Bacillus macerans enzyme were determined in 2mM sodium cacodylate pH 6.0,1.5 M guanidine hydrochloride, containing 1 mM CaCl₂ or 1 mM EDTA Melting of H(A12-M)ΔY13 and H(A16-M) in the presence of calcium ions is characterized by two subtransitions; only one transition is observed in the case of H(A12-M). In calcium-free buffer each of the three hybrid enzymes melts in one two-state transition. Transition temperatures T _m and molar enthalpy changes ΔH are reduced in the absence of calcium ions but the reduction is much more pronounced for H(A12-M)ΔY13 and H(A16-M) than for the less thermostable enzyme H(A12-M).     

Procoagulant Platelets Form an α-Granule Protein-covered “Cap” on Their Surface That Promotes Their Attachment to Aggregates

Strongly activated “coated” platelets are characterized by increased phosphatidylserine (PS) surface expression, α-granule protein retention, and lack of active integrin α_IIbβ₃. To study how they are incorporated into thrombi despite a lack of free activated integrin, we investigated the structure, function, and formation of the α-granule protein “coat.” Confocal microscopy revealed that fibrin(ogen) and thrombospondin colocalized as “cap,” a single patch on the PS-positive platelet surface. In aggregates, the cap was located at the point of attachment of the PS-positive platelets. Without fibrin(ogen) retention, their ability to be incorporated in aggregates was drastically reduced. The surface fibrin(ogen) was strongly decreased in the presence of a fibrin polymerization inhibitor GPRP and also in platelets from a patient with dysfibrinogenemia and a fibrinogen polymerization defect. In contrast, a fibrinogen-clotting protease ancistron increased the amount of fibrin(ogen) and thrombospondin on the surface of the PS-positive platelets stimulated with collagen-related peptide. Transglutaminases are also involved in fibrin(ogen) retention. However, platelets from patients with factor XIII deficiency had normal retention, and a pan-transglutaminase inhibitor T101 had only a modest inhibitory effect. Fibrin(ogen) retention was normal in Bernard-Soulier syndrome and kindlin-3 deficiency, but not in Glanzmann thrombasthenia lacking the platelet pool of fibrinogen and α_IIbβ₃. These data show that the fibrin(ogen)-covered cap, predominantly formed as a result of fibrin polymerization, is a critical mechanism that allows coated (or rather “capped”) platelets to become incorporated into thrombi despite their lack of active integrins.     

Chitinase 1 Is a Biomarker for and Therapeutic Target in Scleroderma-Associated Interstitial Lung Disease That Augments TGF-β1 Signaling

Interstitial lung disease (ILD) with pulmonary fibrosis is an important manifestation in systemic sclerosis (SSc, scleroderma) where it portends a poor prognosis. However, biomarkers that predict the development and or severity of SSc-ILD have not been validated, and the pathogenetic mechanisms that engender this pulmonary response are poorly understood. In this study, we demonstrate in two different patient cohorts that the levels of chitotriosidase (Chit1) bioactivity and protein are significantly increased in the circulation and lungs of SSc patients compared with demographically matched controls. We also demonstrate that, compared with patients without lung involvement, patients with ILD show high levels of circulating Chit1 activity that correlate with disease severity. Murine modeling shows that in comparison with wild-type mice, bleomycin-induced pulmonary fibrosis was significantly reduced in Chit1(-/-) mice and significantly enhanced in lungs from Chit1 overexpressing transgenic animals. In vitro studies also demonstrated that Chit1 interacts with TGF-β1 to augment fibroblast TGF-β receptors 1 and 2 expression and TGF-β-induced Smad and MAPK/ERK activation. These studies indicate that Chit1 is potential biomarker for ILD in SSc and a therapeutic target in SSc-associated lung fibrosis and demonstrate that Chit1 augments TGF-β1 effects by increasing receptor expression and canonical and noncanonical TGF-β1 signaling.     

15N-Harnstoffumsatz bei Schafen nach Verabreichung in gelöster Form

Albinoratten (Wistar) mit einer Lebendmasse von etwa 100 g erhielten in 26 Versuchsgruppen (4 Tiere/Gruppe) verschiedene Diäten in denen der Lysinbedarf durch die Diät bzw. Lysinzulagen zu 75 %, 100 % bzw. 125 % gedeckt wurde. Hierbei handelte es sich um die Diäten auf der Grundlage von Gerste (G), Weizen (W), Weizengluten (WG), isoliertes Sojaprotein (S) und Sojaextraktionsschrot (SE). Für WG und S konnten nur die Lysinstufen 100 % und 125 % und für SE nur 116 % und 125 % realisiert werden. Alle Versuchsgruppen wurden ohne Antibiotikazulage und mit Antibiotikazulage in Form von 7 g Nebacitin/kg Futter‐TS über 10 Tage gefüttert. Während der 7tägigen Hauptperiode wurde den Diäten 0, 5 g ^I5N‐Lysin/kg TS zugesetzt (48,3 Atom‐% ¹⁵N‐Überschuß, α‐Aminogruppe zu 95 % ¹⁵N‐markiert).

Die N‐Bilanz konnte nur im Falle von S 100 durch die Antibiotikazulage verbessert werden. Es wird vermutet, daß die 2. limitierende Aminosäure Methionin durch Nebacitin im Darmtrakt vor mikrobiellem Abbau geschützt wurde.

Die biologische Wertigkeit der Futterproteine verschlechterte sich im Falle von G und W durch die Antibiotikazulage, da der resorbierte N‐Anteil erhöht wurde, die Bezugsbasis dadurch größer wurde ohne eine Verbesserung der N‐Verwertung zu erzielen.

Die ¹³N‐Ausscheidung über den Kot wurde im Falle der Diäten G, W und WG durch die Antibiotikazulage signifikant erniedrigt und die ¹⁵N‐Ausscheidung über den Harn in den meisten Fällen erhöht.

Die Ermittlung einer Bruttoverwertung von Lysin bzw. ^I5N‐Lysin, die eine Beziehung zum Lysinansatz (availability) hat, ist auch mit Hilfe einer Markierung der Diät mit ¹⁵N‐markiertem Lysin nicht möglich.     

Structures of SHV-1 β-Lactamase with Penem and Penam Sulfone Inhibitors That Form Cyclic Intermediates Stabilized by Carbonyl Conjugation

Bacterial β-lactamase enzymes are in large part responsible for the decreased ability of β-lactam antibiotics to combat infections. The inability to overcome β-lactamase mediated resistance spurred the development of inhibitors with penems and penam sulfones being amongst the most potent and broad spectrum mechanism-based inactivators. These inhibitors form covalent, “suicide-type” inhibitory intermediates that are attached to the catalytic S70 residue. To further probe the details of the mechanism of β-lactamase inhibition by these novel compounds, we determined the crystal structures of SHV-1 bound with penem 1, and penam sulfones SA1-204 and SA3-53. Comparison with each other and with previously determined crystal structures of members of these classes of inhibitors suggests that the final conformation of the covalent adduct can vary greatly amongst the complex structures. In contrast, a common theme of carbonyl conjugation as a mechanism to avoid deacylation emerges despite that the penem and penam sulfone inhibitors form different types of intermediates. The detailed insights gained from this study could be used to further improve new mechanism-based inhibitors of these common class A serine β-lactamases.