Old role with new feature: T2SS ATPase as a cyclic-di-GMP receptor to regulate antibiotic production.

Cyclic di-GMP (c-di-GMP) is a crucial signaling molecule found extensively in bacteria, involved in the regulation of various physiological and biochemical processes such as biofilm formation, motility, and pathogenicity through binding to downstream receptors. However, the structural dissimilarity of c-di-GMP receptor proteins has hindered the discovery of many such proteins. In this study, we identified LspE, a homologous protein of the type II secretion system (T2SS) ATPase GspE in Lysobacter enzymogenes, as a receptor protein for c-di-GMP. We identified the more conservative c-di-GMP binding amino acid residues as K358 and T359, which differ from the previous reports, indicating that GspE proteins may represent a class of c-di-GMP receptor proteins. Additionally, we found that LspE in L. enzymogenes also possesses a novel role in regulating the production of the antifungal antibiotic HSAF. Further investigations revealed the critical involvement of both ATPase activity and c-di-GMP binding in LspE-mediated regulation of HSAF (Heat-Stable Antifungal Factor) production, with c-di-GMP binding having no impact on LspE's ATPase activity. This suggests that the control of HSAF production by LspE encompasses two distinct processes: c-di-GMP binding and the inherent ATPase activity of LspE. Overall, our study unraveled a new function for the conventional protein GspE of the T2SS as a c-di-GMP receptor protein and shed light on its role in regulating antibiotic production.IMPORTANCEThe c-di-GMP signaling pathway in bacteria is highly intricate. The identification and functional characterization of novel receptor proteins have posed a significant challenge in c-di-GMP research. The type II secretion system (T2SS) is a well-studied secretion system in bacteria. In this study, our findings revealed the ATPase GspE protein of the T2SS as a class of c-di-GMP receptor protein. Notably, we discovered its novel function in regulating the production of antifungal antibiotic HSAF in Lysobacter enzymogenes. Given that GspE may be a conserved c-di-GMP receptor protein, it is worthwhile for researchers to reevaluate its functional roles and mechanisms across diverse bacterial species.

LAM-1 from Lysobacter antibioticus: A potent zinc-dependent activity that inactivates ß-lactam antibiotics.

Resistance to ß-lactam antibiotics, including the "last-resort" carbapenems, has emerged as a major threat to global health. A major resistance mechanism employed by pathogens involves the use of metallo-ß-lactamases (MBLs), zinc-dependent enzymes that inactivate most of the ß-lactam antibiotics used to treat infections. Variants of MBLs are frequently discovered in clinical environments. However, an increasing number of such enzymes have been identified in microorganisms that are less impacted by human activities. Here, an MBL from Lysobacter antibioticus, isolated from the rhizosphere, has been shown to be highly active toward numerous ß-lactam antibiotics. Its activity is higher than that of some of the most effective MBLs linked to hospital-acquired antibiotic resistance and thus poses an interesting system to investigate evolutionary pressures that drive the emergence of such biocatalysts.

Cloning, expression, and characterization of a GH 19-type chitinase with antifungal activity from Lysobacter sp. MK9-1.

The chitin-assimilating gram-negative bacterium, Lysobacter sp. MK9-1, was isolated from soil and was the source of a glycoside hydrolase family 19-type chitinase (Chi19MK) gene that is 933-bp long and encodes a 311-residue protein. The deduced amino acid sequence of Chi19MK includes a signal peptide, an uncharacterized sequence, a carbohydrate-binding module family 12-type chitin binding domain, and a catalytic domain. The catalytic domain of Chi19MK is approximately 60% similar to those of ChiB from Burkholderia gladioli CHB101, chitinase N (ChiN) from Chitiniphilus shinanonensis SAY3T, ChiF from Streptomyces coelicolor A3(2), Chi30 from Streptomyces olivaceoviridisis, ChiA from Streptomyces cyaneus SP-27, and ChiC from Streptomyces griseus HUT6037. Chi19MK lacking the signal and uncharacterized sequences (Chi19MKΔNTerm) was expressed in Escherichia coli Rosetta-gami B(DE3), resulting in significant chitinase activity in the soluble fraction. Purified Chi19MKΔNTerm hydrolyzed colloidal chitin and released disaccharide. Furthermore, Chi19MKΔNTerm inhibited hyphal extension in Trichoderma reesei and Schizophyllum commune. Based on quantitative antifungal activity assays, Chi19MKΔNTerm inhibits the growth of Trichoderma viride with an IC50 value of 0.81 µM.

Lytic potential of Lysobacter capsici VKM B-2533T: bacteriolytic enzymes and outer membrane vesicles.

Recent recurrent outbreaks of bacterial resistance to antibiotics have shown the critical need to identify new lytic agents to combat them. The species Lysobacter capsici VKM B-2533T possesses a potent antimicrobial action against a number of bacteria, fungi and yeasts. Its activity can be due to the impact of bacteriolytic enzymes, antibiotics and peptides. This work isolated four homogeneous bacteriolytic enzymes and a mixture of two proteins, which also had a bacteriolytic activity. The isolates included proteins identical to L. enzymogenes α- and ß-lytic proteases and lysine-specific protease. The proteases of 26 kDa and 29 kDa and a protein identified as N-acetylglycosaminidase had not been isolated in Lysobacter earlier. The isolated ß-lytic protease digested live methicillin-resistant staphylococcal cells with high efficiency (minimal inhibitory concentration, 2.85 µg/mL). This property makes the enzyme deserving special attention. A recombinant ß-lytic protease was produced. The antimicrobial potential of the bacterium was contributed to by outer membrane vesicles (OMVs). L. capsici cells were found to form a group of OMVs responsible for antifungal activity. The data are indicative of a significant antimicrobial potential of this bacterium that requires thorough research.

Cell permeability, ß-lactamase activity, and transport contribute to high level of resistance to ampicillin in Lysobacter enzymogenes.

Discovery of multidrug resistance (MDR) in environmental microorganisms provides unique resources for uncovering antibiotic resistomes, which could be vital to predict future emergence of MDR pathogens. Our previous studies indicated that Lysobacter sp. conferred intrinsic resistance to multiple antibiotics at high levels, especially ampicillin, the first broad-spectrum ß-lactam antibiotics against both Gram-positive and Gram-negative bacteria. However, the underlying molecular mechanisms for resistance to ampicillin in Lysobacter enzymogenes strain C3 (LeC3) remain unknown. In this study, screening a Tn5 transposon mutant library of LeC3 recovered 12 mutants with decreased ampicillin resistance, and three mutants (i.e., tatC, lebla, and lpp) were selected for further characterization. Our results revealed that genes encoding ß-lactamase (lebla) and twin-arginine translocation (tatC) system for ß-lactamase transport played a pivotal role in conferring ampicillin resistance in L. enzymogenes. It was also demonstrated that the lpp gene was not only involved in resistance against ß-lactams but also conferred resistance to multiple antibiotics in L. enzymogenes. Permeability assay results indicated that decreased MDR in the lpp mutant was in part due to its higher cellular permeability. Furthermore, our results showed that the difference of LeC3 and L. antibioticus strain LaATCC29479 in ampicillin susceptibility was partly due to their differences in cellular permeability, but not due to ß-lactamase activities.

A novel heterologous expression strategy for the quorum-quenching enzyme MomL in Lysobacter enzymogenes to the inhibit pathogenicity of Pectobacterium.

Quorum-quenching (QQ) enzymes can block the quorum-sensing (QS) system and prevent the expression of QS-controlled pathogenic factors in bacteria. However, the low expression levels of QQ proteins in the original host bacteria have affected their widespread application. In this study, we heterologously expressed momL, encoding a QQ enzyme with high activity, in Lysobacter enzymogenes. A "yellow-to-white" selection marker and the high-constitutive-expression promoter PgroEL were used in this novel heterologous expression system. In addition, we optimized the spacer between the SD sequence and the initiator to improve the efficiency of the expression system by 1.54-fold. The engineered strain LeMomL degraded the AHL molecule and the virulence factors of Pectobacterium carotovorum subsp. carotovora (Pcc). Additionally, LeMomL significantly decreased the disease caused by Pcc in Chinese cabbages and carrot root tissues. In conclusion, this novel and facile L. enzymogenes expression strategy has good prospects and is an ideal approach for foreign protein expression.

Two forms of phosphomannomutase in gammaproteobacteria: The overlooked membrane-bound form of AlgC is required for twitching motility of Lysobacter enzymogenes.

Lysobacter enzymogenes, a member of Xanthomonadaceae, is a promising tool to control crop-destroying fungal pathogens. One of its key antifungal virulence factors is the type IV pili that are required for twitching motility. Transposon mutagenesis of L. enzymogenes revealed that the production of type IV pili required the presence of the Le2152 gene, which encodes an AlgC-type phosphomannomutase/phosphoglucomutase (PMM). However, in addition to the cytoplasmic PMM domain, the Le2152 gene product contains a ~200-aa N-terminal periplasmic domain that is anchored in the membrane by two transmembrane segments and belongs to the dCache superfamily of periplasmic sensor domains. Sequence analysis identified similar membrane-anchored PMMs, encoded in conserved coaBC-dut-algC gene clusters, in a variety of gammaproteobacteria, either as the sole PMM gene in the entire genome or in addition to the gene encoding the stand-alone enzymatic domain. Previously overlooked N-terminal periplasmic sensor domains were detected in the well-characterized PMMs of Pseudomonas aeruginosa and Xanthomonas campestris, albeit not in the enzymes from Pseudomonas fluorescens, Pseudomonas putida or Azotobacter vinelandii. It appears that after the initial cloning of the enzymatically active soluble part of P. aeruginosa AlgC in 1991, all subsequent studies utilized N-terminally truncated open reading frames. The N-terminal dCache sensor domain of AlgC is predicted to modulate the PMM activity of the cytoplasmic domain in response to as yet unidentified environmental signal(s). AlgC-like membrane-bound PMMs appear to comprise yet another environmental signalling system that regulates the production of type IV pili and potentially other systems in certain gammaproteobacteria.

Structural and functional properties of antimicrobial protein L5 of Lysоbacter sp. XL1.

The Gram-negative bacterium Lysobacter sp. XL1 secretes into the extracellular space five bacteriolytic enzymes that lyse the cell walls of competing microorganisms. Of special interest are homologous lytic proteases L1 and L5. This work found protein L5 to possess Gly-Gly endopeptidase and N-acetylmuramoyl-L-Ala amidase activities with respect to staphylococcal peptidoglycan. Protein L5 was found to be capable of aggregating into amyloid-like fibril structures. The crystal structure of protein L5 was determined at a 1.60-Å resolution. Protein L5 was shown to have a rather high structural identity with bacteriolytic protease L1 of Lysobacter sp. XL1 and α-lytic protease of Lysobacter enzymogenes at a rather low identity of their amino acid sequences. Still, the structure of protein L5 was revealed to have regions that differed from their equivalents in the homologs. The revealed structural distinctions in L5 are suggested to be of importance in exhibiting its unique properties.

ACC Deaminase from Lysobacter gummosus OH17 Can Promote Root Growth in Oryza sativa Nipponbare Plants.

Although Lysobacter species are a remarkable source of natural compounds with antibacterial and antifungal activities, the ability of these bacteria to produce plant growth promoters remains practically unknown. In this work, the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) has been isolated from the secretions of Lysobacter gummosus OH17, indicating the presence of an ACC deaminase, which was shown to be encoded in the gene peg_1256. The recombinant enzyme could not only deaminate ACC to provide 2-oxobutanoic acid but also catalyzed the amination of the 2-oxobutanoic acid, demonstrating, for the first time, that ACC deaminases can produce ACC. After the treatment of rice Oryza sativa Nipponbare plants with OH17 ACC deaminase, the ethylene production levels were 44% higher in comparison with the control experiments, allowing significant improvements in root, 10%, and stem, 14%, growth.

Functional and Structural Analysis of Phenazine O-Methyltransferase LaPhzM from Lysobacter antibioticus OH13 and One-Pot Enzymatic Synthesis of the Antibiotic Myxin.

Myxin is a well-known antibiotic that had been used for decades. It belongs to the phenazine natural products that exhibit various biological activities, which are often dictated by the decorating groups on the heteroaromatic three-ring system. The three rings of myxin carry a number of decorations, including an unusual aromatic N5, N10-dioxide. We previously showed that phenazine 1,6-dicarboxylic acid (PDC) is the direct precursor of myxin, and two redox enzymes (LaPhzS and LaPhzNO1) catalyze the decarboxylative hydroxylation and aromatic N-oxidations of PDC to produce iodinin (1.6-dihydroxy- N5, N10-dioxide phenazine). In this work, we identified the LaPhzM gene from Lysobacter antibioticus OH13 and demonstrated that LaPhzM encodes a SAM-dependent O-methyltransferase converting iodinin to myxin. The results further showed that LaPhzM is responsible for both monomethoxy and dimethoxy formation in all phenazine compounds isolated from strain OH13. LaPhzM exhibits relaxed substrate selectivity, catalyzing O-methylation of phenazines with non-, mono-, or di- N-oxide. In addition, we demonstrated a one-pot biosynthesis of myxin by in vitro reconstitution of the three phenazine-ring decorating enzymes. Finally, we determined the X-ray crystal structure of LaPhzM with a bound cofactor at 1.4 Å resolution. The structure provided molecular insights into the activity and selectivity of the first characterized phenazine O-methyltransferase. These results will facilitate future exploitation of the thousands of phenazines as new antibiotics through metabolic engineering and chemoenzymatic syntheses.

A new cold-adapted serine peptidase from Antarctic Lysobacter sp. A03: Insights about enzyme activity at low temperatures.

Currently, there is a great interest for customized biocatalysts that can supply the ongoing demand of industrial processes, but also deal with the growing concern about the environment. In this scenario, cold-adapted enzymes have features that make them very attractive for industrial and biotechnological purposes. Here, we describe A03Pep1, a new cold-adapted serine peptidase isolated from Lysobacter sp. A03 by screening a genomic library. The enzyme is synthesized as a large inactive prepropeptidase that, after intramolecular processing, gives rise to the active form, of 35kDa. The heterologous expression of A03Pep1 was carried out in E. coli cells harboring the vector pGEX-4T-2-a0301. Its activity was optimal at pH 9.0 and 40°C, in the presence of 25mM Ca2+, which may contribute to the thermal stability of the enzyme. The 3D structure modelling predicted a less deep and more open binding pocket in A03Pep1 than that observed in the crystal structure of its mesophilic homologous AprV2, presumably as a way to enhance the probability of substrate binding at low temperatures. These results provide possible approaches in developing new biotechnologically relevant peptidases active at low to moderate temperatures.

Direct Regulation of Extracellular Chitinase Production by the Transcription Factor LeClp in Lysobacter enzymogenes OH11.

Lysobacter enzymogenes is a gram-negative bacterial biological control agent that produces abundant extracellular enzymes capable of degrading the cell walls of fungal pathogens. In strain OH11, an isolate from China, the global regulator LeClp controls the production of extracellular chitinase by regulating the transcription of the chitinase-encoding gene chiA. Using a combination of bioinformatic, genetic, and biochemical methods, we show that LeClp regulates chiA transcription by directly binding to the chiA promoter region. Although LeClp appears to be important in this role, it is not the sole regulator of chiA transcription. Furthermore, the sequence analysis of putative LeClp binding sites indicated that the LeClp homolog could be involved in the regulation of extracellular chitinase production in diverse Lysobacter spp. by a mechanism similar to that in L. enzymogenes. Our findings present new insights into the molecular mechanism of LeClp in controlling extracellular chitinase activity, providing a fundamental road to elucidate how LeClp regulates the production of other extracellular lytic enzymes in L. enzymogenes.

Heterocyclic Aromatic N-Oxidation in the Biosynthesis of Phenazine Antibiotics from Lysobacter antibioticus.

Heterocyclic aromatic N-oxides often have potent biological activities, but the mechanism for aromatic N-oxidation is unclear. Six phenazine antibiotics were isolated from Lysobacter antibioticus OH13. A 10 gene cluster was identified for phenazine biosynthesis. Mutation of LaPhzNO1 abolished all N-oxides, while non-oxides markedly increased. LaPhzNO1 is homologous to Baeyer-Villiger flavoproteins but was shown to catazlye phenazine N-oxidation. LaPhzNO1 and LaPhzS together converted phenazine 1,6-dicarboxylic acid to 1,6-dihydroxyphenazine N5,N10-dioxide. LaPhzNO1 also catalyzed N-oxidation of 8-hydroxyquinoline.

Outer membrane vesicles of Lysobacter sp. XL1: biogenesis, functions, and applied prospects.

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have been intensively investigated in recent times. Vesicle formation models have been proposed, some factors affecting the process were established, and important roles vesicles play in vital activities of their producing cells were determined. Studies of pathogenic bacterial vesicles contribute to understanding the causes of acute infection and developing drugs on their basis. Despite intensive research, issues associated with the understanding of vesicle biogenesis, the mechanisms of bacterium-bacterium and pathogen-host interactions with participation of vesicles, still remain unresolved. This review discusses some results obtained in the research into OMVs of Lysobacter sp. XL1 VKM B-1576. This bacterium secretes into the environment a spectrum of bacteriolytic enzymes that hydrolyze peptidoglycan of competing bacteria, thus leading to their lysis. One of these enzymes, lytic endopeptidase L5, has been shown not only to be secreted by means of vesicles but also to be involved in their formation. As part of vesicles, the antimicrobial potential of L5 enzyme has been found to be considerably expanded. Vesicles have been shown to have a therapeutic effect in respect of anthrax infection and staphylococcal sepsis modelled in mice. The scientific basis for constructing liposomal antimicrobial preparations from vesicle phospholipids and recombinant bacteriolytic enzyme L5 has been formed.

Structural Studies of Component of Lysoamidase Bacteriolytic Complex from Lysobacter sp. XL1.

The lysoamidase bacteriolytic complex (LBC) comprising five enzymes (L1-L5) is secreted into the culture liquid by gram-negative bacterium Lysobacter sp. XL1. The medicinal agent lysoamidase has a broad-antimicrobial spectrum. Bacteriolytic protease L1 belongs to the LBC. Recombinant L1 protease of Lysobacter sp. XL1 was expressed, purified to homogeneity and crystallized. The X-ray structure of L1 at 1.35 Å resolution has been determined using the synchrotron data and the molecular replacement method. L1 protease is a thermostable whose thermal unfolding proceeds in one step without forming stable intermediates. Structural information concerning L1 will contribute to the development of new-generation antimicrobial drugs, whose application will not be accompanied by the selection of resistant microorganisms.

Heterologous expression and pro-peptide supported refolding of the high specific endopeptidase Lys-C.

The high specific lysyl endopeptidase (Lys-C; EC 3.4.21.50) is often used for the initial fragmentation of polypeptide chains during protein sequence analysis. However, due to its specificity it could be a useful tool for the production of tailor-made protein hydrolysates with for example bioactive or techno functional properties. Up to now, the high price makes this application nearly impossible. In this work, the increased expression for Escherichia coli optimized Lys-C was investigated. The cloned sequence had a short artificial N-terminal pro-peptide (MGSK). The expression of MGSK-Lys-C was tested using three expression vectors and five E. coli host strains. The highest expression rate was obtained for the expression system consisting of the host strain E. coli JM109 and the rhamnose inducible expression vector pJOE. A Lys-C activity of 9340 ± 555 nkatTos-GPK-pNA/Lculture could be achieved under optimized cultivation conditions after chemical refolding. Furthermore, the influence of the native pre-N-pro peptide of Lys-C from Lysobacter enzymogenes ssp. enzymogenes ATCC 27796 on Lys-C refolding was investigated. The pre-N-pro peptide was expressed recombinantly in E. coli JM109 using the pJOE expression vector. The optimal concentration of the pre-N-pro peptide in the refolding procedure was 100 µg/mLrefolding buffer and the Lys-C activity could be increased to 541,720 nkatTos-GPK-pNA/Lculture. With the results presented, the expensive lysyl endopeptidase can be produced in high activity and high amounts and the potential of Lys-C for tailor-made protein hydrolysates with bioactive (e.g. antihypertensive) and/or techno functional (e.g. foaming, emulsifying) properties can be investigated in future time studies.

Gene Expression of Lytic Endopeptidases AlpA and AlpB from Lysobacter sp. XL1 in Pseudomonads.

Development of an efficient expression system for (especially secreted) bacterial lytic enzymes is a complicated task due to the specificity of their action. The substrate for such enzymes is peptidoglycan, the main structural component of bacterial cell walls. For this reason, expression of recombinant lytic proteins is often accompanied with lysis of the producing bacterium. This paper presents data on the construction of an inducible system for expression of the lytic peptidases AlpA and AlpB from Lysobacter sp. XL1 in Pseudomonas fluorescens Q2-87, which provides for the successful secretion of these proteins into the culture liquid. In this system, the endopeptidase gene under control of the T7lac promoter was integrated into the bacterial chromosome, as well as the Escherichia coli lactose operon repressor protein gene. The T7 pol gene under lac promoter control, which encodes the phage T7 RNA polymerase, is maintained in Pseudomonas cells on the plasmids. Media and cultivation conditions for the recombinant strains were selected to enable the production of AlpA and AlpB by a simple purification protocol. Production of recombinant lytic enzymes should contribute to the development of new-generation antimicrobial drugs whose application will not be accompanied by selection of resistant microorganisms.

Identification of a small molecule signaling factor that regulates the biosynthesis of the antifungal polycyclic tetramate macrolactam HSAF in Lysobacter enzymogenes.

Lysobacter species are emerging as new sources of antibiotics. The regulation of these antibiotics is not well understood. Here, we identified a small molecule metabolite (LeDSF3) that regulates the biosynthesis of the antifungal antibiotic heat-stable antifungal factor (HSAF), a polycyclic tetramate macrolactam with a structure and mode of action distinct from the existing antifungal drugs. LeDSF3 was isolated from the culture broth of Lysobacter enzymogenes, and its chemical structure was established by NMR and MS. The purified compound induced green fluorescence in a reporter strain of Xanthomonas campestris, which contained a gfp gene under the control of a diffusible signaling factor (DSF)-inducible promoter. Exogenous addition of LeDSF3 in L. enzymogenes cultures significantly increased the HSAF yield, the transcription of HSAF biosynthetic genes, and the antifungal activity of the organism. The LeDSF3-regulated HSAF production is dependent on the two-component regulatory system RpfC/RpfG. Moreover, LeDSF3 upregulated the expression of the global regulator cAMP receptor-like protein (Clp). The disruption of clp led to no HSAF production. Together, the results show that LeDSF3 is a fatty acid-derived, diffusible signaling factor positively regulating HSAF biosynthesis and that the signaling is mediated by the RfpC/RpfG-Clp pathway. These findings may facilitate the antibiotic production through applied genetics and molecular biotechnology in Lysobacter, a group of ubiquitous yet underexplored microorganisms.

Characterization of water-soluble dark-brown pigment from Antarctic bacterium, Lysobacter oligotrophicus.

Lysobacter oligotrophicus strain 107-E2(T) isolated from Antarctica produces dark-brown colored water-soluble pigment, in addition to hydrolases and lytic enzymes. The production of pigment is a common characteristic among members of the genus Lysobacter, but the identity of the pigments has been unknown. In this study, we identified the pigment from L. oligotrophicus as melanin pigment (Lo-melanin) by chemical and spectroscopic analyses. Although melanin is generally insoluble in both aqueous and organic solvents, the results in this study revealed that Lo-melanin shows water-solubility by means of the added polysaccharide chain. Lo-melanin production of L. oligotrophicus was increased by ultraviolet (UV) exposure, and survival rate of Escherichia coli under UV-irradiated condition was increased by the addition of Lo-melanin to the medium.

Atomic resolution structure of a lysine-specific endoproteinase from Lysobacter enzymogenes suggests a hydroxyl group bound to the oxyanion hole.

Lysobacter enzymogenes lysyl endoproteinase (LysC) is a trypsin-type serine protease with a high pH optimum that hydrolyses all Lys-Xaa peptide bonds. The high specificity of LysC renders it useful for biotechnological purposes. The K30R variant of a related lysyl endoproteinase from Achromobacter lyticus has favourable enzymatic properties that might be transferrable to LysC. To visualize structural differences in the substrate-binding sites, the crystal structures of wild-type and the K30R variant of LysC were determined. The mutation is located at a distance of 12 Šfrom the catalytic triad and subtly changes the surface properties of the substrate-binding site. The high pH optimum of LysC can be attributed to electrostatic effects of an aromatic Tyr/His stack on the catalytic aspartate and is a general feature of this enzyme subfamily. LysC crystals in complex with the covalent inhibitor N(α)-p-tosyl-lysyl chloromethylketone yielded data to 1.1 and 0.9 Šresolution, resulting in unprecedented precision of the active and substrate-binding sites for this enzyme subfamily. Error estimates on bond lengths and difference electron density indicate that instead of the expected oxyanion a hydroxyl group binds to the partially solvent-exposed oxyanion hole. Protonation of the alkoxide catalytic intermediate might be a recurring feature during serine protease catalysis.