Parkinson's disease-risk protein TMEM175 is a proton-activated proton channel in lysosomes.

Lysosomes require an acidic lumen between pH 4.5 and 5.0 for effective digestion of macromolecules. This pH optimum is maintained by proton influx produced by the V-ATPase and efflux through an unidentified "H+ leak" pathway. Here we show that TMEM175, a genetic risk factor for Parkinson's disease (PD), mediates the lysosomal H+ leak by acting as a proton-activated, proton-selective channel on the lysosomal membrane (LyPAP). Acidification beyond the normal range potently activated LyPAP to terminate further acidification of lysosomes. An endogenous polyunsaturated fatty acid and synthetic agonists also activated TMEM175 to trigger lysosomal proton release. TMEM175 deficiency caused lysosomal over-acidification, impaired proteolytic activity, and facilitated α-synuclein aggregation in vivo. Mutational and pH normalization analyses indicated that the channel's H+ conductance is essential for normal lysosome function. Thus, modulation of LyPAP by cellular cues may dynamically tune the pH optima of endosomes and lysosomes to regulate lysosomal degradation and PD pathology.

In search for structural targets for engineering d-amino acid transaminase: modulation of pH optimum and substrate specificity.

The development of biocatalysts requires reorganization of the enzyme's active site to facilitate the productive binding of the target substrate and improve turnover number at desired conditions. Pyridoxal-5'-phosphate (PLP) - dependent transaminases are highly efficient biocatalysts for asymmetric amination of ketones and keto acids. However, transaminases, being stereoselective enzymes, have a narrow substrate specificity due to the ordered structure of the active site and work only in neutral-alkaline media. Here, we investigated the d-amino acid transaminase from Aminobacterium colombiense, with the active site organized differently from that of the canonical d-amino acid transaminase from Bacillus sp. YM-1. Using a combination of site-directed mutagenesis, kinetic analysis, molecular modeling, and structural analysis we determined the active site residues responsible for substrate binding, substrate differentiation, thermostability of a functional dimer, and affecting the pH optimum. We demonstrated that the high specificity toward d-glutamate/α-ketoglutarate is due to the interactions of a γ-carboxylate group with K237 residue, while binding of other substrates stems from the effectiveness of their accommodation in the active site optimized for d-glutamate/α-ketoglutarate binding. Furthermore, we showed that the K237A substitution shifts the catalytic activity optimum to acidic pH. Our findings are useful for achieving target substrate specificity and demonstrate the potential for developing and optimizing transaminases for various applications.

Engineering the optimum pH of ß-galactosidase from Aspergillus oryzae for efficient hydrolysis of lactose.

ß-Galactosidase (lacA) from Aspergillus oryzae is widely used in the dairy industry. Its acidic pH optimum and severe product inhibition limit its application for lactose hydrolysis in milk. In the present study, structure-based sequence alignment was conducted to determine the candidate mutations to shift the pH optimum of lacA to the neutral range. The Y138F and Y364F mutants shifted the pH optimum of lacA from 4.5 to 5.5 and 6.0, respectively. The acid dissociation constant (pKa) values of catalytic acid/base residues with upwards shift were consistent with the increased pH optimum. All variants in the present study also alleviated galactose inhibition to various extents. Molecular dynamics demonstrated that the less rigid tertiary structures and lower galactose-binding free energy of Y138F and Y364F might facilitate the release of the end product. Both Y138F and Y364F mutants exhibited better hydrolytic ability than lacA in milk lactose hydrolysis. The higher pH optimum and lower galactose inhibition of Y138F and Y364F may explain their superiority over lacA. The Y138F and Y364F mutants in the present study showed potential in producing low-lactose milk, and our studies provide a novel strategy for engineering the pH optimum of glycoside hydrolase.

Shifting the pH Optima of (R)-Selective Transaminases by Protein Engineering.

Amine transaminases (ATAs) are powerful biocatalysts for the stereoselective synthesis of chiral amines. However, wild-type ATAs usually show pH optima at slightly alkaline values and exhibit low catalytic activity under physiological conditions. For efficient asymmetric synthesis ATAs are commonly used in combination with lactate dehydrogenase (LDH, optimal pH: 7.5) and glucose dehydrogenase (GDH, optimal pH: 7.75) to shift the equilibrium towards the synthesis of the target chiral amine and hence their pH optima should fit to each other. Based on a protein structure alignment, variants of (R)-selective transaminases were rationally designed, produced in E. coli, purified and subjected to biochemical characterization. This resulted in the discovery of the variant E49Q of the ATA from Aspergillus fumigatus, for which the pH optimum was successfully shifted from pH 8.5 to 7.5 and this variant furthermore had a two times higher specific activity than the wild-type protein at pH 7.5. A possible mechanism for this shift of the optimal pH is proposed. Asymmetric synthesis of (R)-1-phenylethylamine from acetophenone in combination with LDH and GDH confirmed that the variant E49Q shows superior performance at pH 7.5 compared to the wild-type enzyme.

Thermostable arginase from Sulfobacillus acidophilus with neutral pH optimum applied for high-efficiency L-ornithine production.

This study aims to use neutral pH optimum arginase as the catalyst for high-efficiency L-ornithine production. Sulfobacillus acidophilus arginase was firstly cloned and overexpressed in Escherichia coli. The purified enzyme was obtained, and the molecular mass determination showed that this arginase was a hexamer. S. acidophilus arginase possessed similarities with the other arginases such as the conserved sequences, purification behavior, and the necessity for Mn2+ as a cofactor. The maximum enzyme activity was obtained at pH 7.5 and 70 °C. Thermostability and pH stability analysis showed that the arginase was stable at 30-60 °C and pH 7.0-8.5, respectively. The kinetic parameters suggested that S. acidophilus arginase could efficiently hydrolyze L-arginine. Bioconversion with this neutral pH optimum arginase had the advantages of avoiding producing by-product, high molar yield, and high-level production of L-ornithine. When the bioconversion was performed with a fed-batch strategy and a coupled-enzyme system involving S. acidophilus arginase and Jack bean urease, the final production of 2.87 mol/L was obtained with only 1.72 mmol/L L-arginine residue, and the molar yield was 99.9%. The highest production record suggests that S. acidophilus arginase has a great prospect in industrial L-ornithine production.

Biological properties of influenza A virus mutants with amino acid substitutions in the HA2 glycoprotein of the HA1/HA2 interaction region.

Influenza A viruses (IAVs) enter into cells by receptor-dependent endocytosis. Subsequently, conformational changes of haemagglutinin are triggered by low environmental pH and the N terminus of HA2 glycoprotein (gp) is inserted into the endosomal membrane, resulting in fusion pore formation and genomic vRNA release into the cytoplasm. However, the pH optimum of membrane fusion is host- and virus-specific and can have an impact on virus pathogenicity. We prepared mutants of neurotropic IAV A/WSN/33 (H1N1) with aa substitutions in HA2 gp at the site of HA1/HA2 interaction, namely T642H (HA2 numbering position 64, H1 numbering position HA407; referred to as mutant '64'), V662H ('66') (HA409); and a double mutant ('D') with two aa substitutions (T642H, V662H). These substitutions were hypothesized to influence the pH optimum of fusion. The pH optimum of fusion activity was measured by a luciferase assay and biological properties of viruses were monitored. The in vitro and in vivo replication ability and pathogenicity of mutants were comparable (64) or lower (66, D) than those of the wild-type virus. However, the HA2 mutation V662H and double mutation T642H, V662H shifted the fusion pH maximum to lower values (ranging from 5.1 to 5.3) compared to pH from 5.4 to 5.6 for the wild-type and 64 mutant. The decreased replication ability and pathogenicity of 66 and D mutants was accompanied by higher titres in late intervals post-infection in lungs, and viral RNA in brains compared to wild-type virus-infected mice. These results have implications for understanding the pathogenicity of influenza viruses.

Characteristics of PPT1 and TPP1 enzymes in neuronal ceroid lipofuscinosis (NCL) 1 and 2 by dried blood spots (DBS) and leukocytes and their application to newborn screening.

We first characterized PPT1 and TPP1 enzymes in dried blood spots (DBS), plasma/serum, and leukocytes/lymphocytes using neuronal ceroid lipofuscinosis (NCL) 1 and 2 patients and control subjects. PPT1 enzyme had only one acid form in control DBS, plasma/serum, and leukocytes/lymphocytes and showed deficient activities in these samples from NCL 1 patients. Conversely, TPP1 enzymes in control DBS and leukocytes/lymphocytes consisted of two forms, an acidic form and a neutral form, whereas serum TPP1 enzyme had only a neutral form. In control subjects, the optimal pH of PPT1 enzyme in DBS, plasma/serum, and leukocytes/lymphocytes was 4.5 to 5.0 in the acidic form, whereas TPP1 enzyme in control DBS and leukocytes/lymphocytes was pH 4.5 and 6.5, respectively. In NCL 1 and 2, both PPT1 and TPP1 enzyme activities in DBS, plasma, and leukocytes/lymphocytes were markedly reduced in acidic pH, whereas heterozygotes of NCL 1 and 2 in the acidic form showed intermediate activities between patients and control subjects. In neutral conditions, pH 6.0, the PPT1 enzyme activities in NCL 1 patients showed rather higher residual activities and intermediate activities in heterozygotes in NCL 1, which was probably caused by mutated proteins in three cases with NCL 1 patients. TPP1 enzyme activities at neutral pH 6.5 to 7.0 in DBS and leukocytes/lymphocytes showed higher enzyme activities in NCL 2 patients and heterozygotes. The reason for the increases of neutral TPP1 enzyme activities at pH 6.5 to 7.0 in NCL 2 DBS and leukocytes/lymphocytes, is obscure, but possibly caused by secondary activation of neutral TPP1 enzyme due to the absence of the acidic form. Interestingly, TPP1 activity in serum only consisted of a neutral form, no acidic form, and was not deficient in any NCL 2 patient. Therefore, we can diagnose NCL 1 patients by plasma/serum enzyme assay of PPT1, but not diagnose NCL 2 by serum TPP1 enzyme assay. A pilot study of newborn screening of NCL 1 and 2 has been established by more than 1000 newborn DBS assays. Using this assay system, we will be able to perform newborn screening of NCL 1 and 2 by DBS.

Recombinant broad-range phospholipase C from Listeria monocytogenes exhibits optimal activity at acidic pH.

The broad-range phospholipase C (PLC) from Listeria monocytogenes has been expressed using an intein expression system and characterized. This zinc metalloenzyme, similar to the homologous enzyme from Bacillus cereus, targets a wide range of lipid substrates. With monomeric substrates, the length of the hydrophobic acyl chain has significant impact on enzyme efficiency by affecting substrate affinity (Km). Based on a homology model of the enzyme to the B. cereus protein, several active site residue mutations were generated. While this PLC shares many of the mechanistic characteristics of the B. cereus PLC, a major difference is that the L. monocytogenes enzyme displays an acidic pH optimum regardless of substrate status (monomer, micelle, or vesicle). This unusual behavior might be advantageous for its role in the pathogenicity of L. monocytogenes.

The role of fusion activity of influenza A viruses in their biological properties.

Influenza A viruses (IAVs) cause acute respiratory infections of humans, which are repeated yearly. Human IAV infections are associated with significant morbidity and mortality and therefore they represent a serious health problem. All human IAV strains are originally derived from avian IAVs, which, after their adaptation to humans, can spread in the human population and cause pandemics with more or less severe course of the disease. Presently, however, the potential of avian IAV to infect humans and to cause the disease cannot be predicted. Many studies are therefore focused on factors influencing the virulence and pathogenicity of IAV viruses in a given host. The virus-host interaction starts by virus attachment via the envelope glycoprotein hemagglutinin (HA) to the receptors on the cell surface. In addition to receptor binding, HA mediates also the fusion of viral and endosomal membranes, which follows the virus endocytosis. The fusion potential of HA trimer, primed by proteolytic cleavage, is activated by low pH in endosomes, resulting in HA refolding into the fusion-active form. The HA conformation change is predetermined by its 3-D structure, is pH-dependent, irreversible and strain-specific. The process of fusion activation of IAV hemagglutinin is crucial for virus entry into the cell and for the ability of the virus to replicate in the host. Here we discuss the known data about the characteristics of fusion activation of HA in relation to IAV virulence and pathogenicity.

Novel sialidase from non-pathogenic bacterium Oerskovia paurometabola strain O129.

Bacterial sialidases are enzymes that are involved in a number of vital processes in microorganisms and in their interaction with the host or the environment. Their wide application for scientific and applied purposes requires the search for highly effective and non-pathogenic producers. Here, we report the first description of sialidase from Oerskovia paurometabola. The extracellular enzyme preparation was partially purified. The presence of sialidase was confirmed in native PAGE treated with the fluorogenic substrate 4MU-Neu5Ac. Maximum enzyme activity was registered at 37 °C and in the pH range of 4.0-5.5. The influence of metal ions and EDTA was examined. It was demonstrated that EDTA, Mn2+ and Ba2+ ions inhibit the sialidase activity to different extent, while Cd2+, Fe2+ and Fe3+ have stimulating effect on it. These features are studied for the first time concerning sialidase of Oerskovia representative. Cell bound sialidase and sialate aldolase were also established.

Modulating the pH profile of the pullulanase from Pyrococcus yayanosii CH1 by synergistically engineering the active center and surface.

A preferable pullulanase with high thermostability and catalytic activity at pH 4.5-5 is desired to match with glucoamylase in the starch-saccharification process. However, most of them exhibit low activity under such low pH conditions. Here, the optimal pH of the hyperthermostable pullulanase from Pyrococcus yayanosii (PulPY2) was successfully shifted from 6.4 to 5 with a 2-fold increase in the specific activity based on synergistic engineering of the active center and surface. Synergistic engineering was performed by introducing histidine within 6 Å of the active sites, and by enhancing negative charges on the enzymatic surface. Two single-site mutants of PulPY2-Q13H and PulPY2-I25E with higher hydrolytic activity were obtained, the optimal pH of which was shifted to pH 5 and 5.4, respectively; the combined mutant PulPY2-Q13H/I25E exhibited the optimal pH of 5, 3.2-fold increasing catalytic efficiency at pH 5, and high thermostability compared to PulPY2. These results not only obtained an applicable pullulanase for industrial application, but also provided a strategy for shifting the optimal pH of the enzyme based on synergistic engineering of the active center and surface.

Production of biodiesel by Burkholderia cepacia lipase as a function of process parameters.

Despite the already established route of chemically catalyzed transesterification reaction in biodiesel production, due to some of its shortcomings, biocatalysts such as lipases present a vital alternative. Namely, it was noticed that one of the key shortcomings for the optimization of the enzyme catalyzed biodiesel synthesis process is the information on the lipase activity in the reaction mixture. In addition to making optimization difficult, it also makes it impossible to compare the results of the independent research. This article shows how lipase intended for use in biodiesel synthesis can be easily and accurately characterized and what is the enzyme concentration that enables achievement of the desired level of fatty acid methyl esters (FAME) in the final product mixture. Therefore, this study investigated the effect of two different activity loads of Burkholderia cepacia lipase on the biodiesel synthesis varying the pH and temperature optimal for lipase activity. The optimal lipase pH and temperature were determined by two different enzyme assays: spectrophotometric and titrimetric. The B. cepacia lipase pH optimum differentiated between assays, while the lipase optimally hydrolyzed substrates at 50°C. The analysis of FAME during 24 hr of biodiesel synthesis, at two different enzyme concentrations, pH 7, 8, and 10, and using two different buffers, revealed that the transesterification reaction at optimal pH, 1 hr reaction time and lipase activity load of 250 U per gram of reaction mixture was sufficient to produce more than 99% FAME.

Hydrogen-bond-based protein engineering for the acidic adaptation of Bacillus acidopullulyticus pullulanase.

Pullulanase is a starch-debranching enzyme that is generally employed to efficiently break down starch for the production of high-glucose syrup. Acidic adaptation of pullulanases is of special interest. In this study, we conducted protein engineering to improve the acidic adaptation of Bacillus acidopullulyticus pullulanase (BaPul) and used a hydrogen-bond-based approach to identify promising residues that may change the deprotonation constants (pKa) of the catalytic residues. A total of 19 amino acids were selected for mutation according to the crystal structure of BaPul. The pH optimum of the L627R mutant shifted from 5.0 to 4.0, and its relative activity at pH 4.0 was 117% that of the wide-type enzyme. The improved efficacy of the L627R mutant at pH 4.0 was confirmed by kinetic parameters and pKa prediction. Moreover, the L627R mutant exhibited increased tolerance against acid-mediated denaturation, and its maximum d-glucose content (97.4%) was obtained after 40 h incubation, which is shorter by 10 h compared with the time required by the wide-type enzyme to produce a comparable amount of the monosaccharide. The L627R mutant may be suitable for industrial application because its shortened reaction time translates to reduced energy consumption.

Assaying Fucosidase Activity.

The characterization of a recombinant glycosidase can be done with commercially available substrates, which enable testing of enzyme functionality and determination of linkage specificity. Colorimetric assays with p-nitrophenyl substrates provide a relatively simple and fast way of screening conditions which could affect enzyme activity (buffer, pH, ion dependence, temperature). These substrates are useful for the determination of activity optima and the characterization of basic activity parameters. However, testing for linkage specificity should be performed on more complex sugars presenting a range of different glycosidic bonds and might need more sophisticated methods of analysis. This protocol provides comprehensive instructions on how to perform an initial characterization of your glycosidase using a recombinant α-L-fucosidase as an example.

Rational and Semirational Protein Design.

This mini review gives an overview over different design approaches and methodologies applied in rational and semirational enzyme engineering. The underlying principles for engineering novel activities, enantioselectivity, substrate specificity, stability, and pH optimum are summarized.

A new method to evaluate temperature vs. pH activity profiles for biotechnological relevant enzymes.

BACKGROUND: Glycoside hydrolases are important for various industrial and scientific applications. Determination of their temperature as well as pH optima and range is crucial to evaluate whether an enzyme is suitable for application in a biotechnological process. These basic characteristics of enzymes are generally determined by two separate measurements. However, these lead to a two-dimensional assessment of the pH range at one temperature (and vice versa) and do not allow prediction of the relative enzymatic performance at any pH/temperature combination of interest. In this work, we demonstrate a new method that is based on experimental data and visualizes the relationship among pH, temperature, and activity at a glance in a three-dimensional contour plot. RESULTS: In this study, we present a method to determine the relative activity of an enzyme at 96 different combinations of pH and temperature in parallel. For this purpose, we used a gradient PCR cycler and a citrate-phosphate-based buffer system in microtiter plates. The approach was successfully tested with various substrates and diverse assays for glycoside hydrolases. Furthermore, its applicability was demonstrated for single enzymes using the endoglucanase Cel8A from Clostridium thermocellum as well as the commercially available complex enzyme mixture Celluclast®. Thereby, we developed a fast and adaptable method to determine simultaneously both pH and temperature ranges of enzymes over a wide range of conditions, an easy transformation of the experimental data into a contour plot for visualization, and the necessary controls. With our method, the suitability of an enzyme or enzyme mixture for any chosen combination of temperature and pH can easily be assessed at a glance. CONCLUSIONS: We propose a method that offers significant advantages over commonly used methods to determine the pH and temperature ranges of enzymes. The overall relationship among pH, temperature, and activity is visualized. Our method could be applied to evaluate exactly what conditions have to be met for optimal utilization of an enzyme or enzyme mixture for both lab-scale and industrial processes. Adaptation to other enzymes, including proteases, should be possible and the method may also lead to a platform for additional applications, such as inactivation kinetics analysis.

Characterization of two glycoside hydrolase family 36 α-galactosidases: novel transglycosylation activity, lead-zinc tolerance, alkaline and multiple pH optima, and low-temperature activity.

Two α-galactosidases, AgaAJB07 from Mesorhizobium and AgaAHJG4 from Streptomyces, were expressed in Escherichia coli. Recombinant AgaAJB07 showed a 2.9-fold and 22.6-fold increase in kcat with a concomitant increase of 2.3-fold and 16.3-fold in Km in the presence of 0.5mM ZnSO4 and 30.0mM Pb(CH3COO)2, respectively. Recombinant AgaAHJG4 showed apparent optimal activity at pH 8.0 in McIlvaine or Tris-HCl buffer and 9.5 in glycine-NaOH or HCl-borax-NaOH buffer, retention of 23.6% and 43.2% activity when assayed at 10 and 20°C, respectively, and a half-life of approximately 2min at 50°C. The activation energies for p-nitrophenyl-α-d-galactopyranoside hydrolysis by AgaAJB07 and AgaAHJG4 were 71.9±0.8 and 48.2±2.0kJmol(-1), respectively. Both AgaAJB07 and AgaAHJG4 exhibited transglycosylation activity, but they required different acceptors and produced different compounds. Furthermore, potential factors for alkaline and multiple pH optima and low-temperature adaptations of AgaAHJG4 were presumed.

Purification and biochemical characterization of membrane-bound neutral ceramidase from camel brain (Camelus dromedarius).

Ceramidases cleave the N-acyl linkages of ceramide to generate sphingosine and its subsequent product sphingosine-1-phosphate (S1P). Ceramide and S1P are important bioactive lipids, and ceramidases are important in regulating the availability of these lipids. In this study, we report the purification and characterization of camel brain neutral ceramidase (CBCDase). The novel CBCDase was purified from camel brain using sequential chromatography of DEAE-Sepharose, Phenyl-Sepharose, Superdex, and Mono Q column. The Mono Q fractions containing ceramidase activity were used for enzyme characterization. The purified CBCDase showed a single band corresponding to a molecular weight of ~100 kDa, displaying classical Michaelis-Menten kinetics, with maximum enzymatic activity at pH 7.0. Deglycosylation of the enzyme yields an apparent molecular weight of ~80 kDa. The purified CBCDase was inhibited by Zn(2+) and Cu(2+), while Ca(2+) stimulates the activity. Phosphatidic acid, phosphatidylserine and phosphatidylcholine completely inhibited enzyme activity at low concentrations. Thiol-containing compounds inhibited the CBCDase activity. Among the nucleotides, ADP, UMP, and TMP inhibited the enzyme activity at low concentrations, whereas, ATP inhibited the activity at higher concentrations only. The CBCDase catalysed both ceramide hydrolysis and reverse CDase reactions. For the first time, we have purified to apparent homogeneity of a ~100 kDa nCDase from camel brain.

Engineering the pH-optimum of activity of the GH12 family endoglucanase by site-directed mutagenesis.

Endo-1,4-ß-glucanase from Penicillium verruculosum (PvEGIII) belongs to family 12 of glycoside hydrolases (GH12). Analysis of the enzyme 3D model structure showed that the amino acid residue Asp98 may directly affect the pH-profile of enzyme activity since it is located at the distance of hydrogen bond formation from Glu203 that plays the role of a general acid in catalysis. The gene encoding the PvEGIII was cloned into Escherichia coli. After the deletion of two introns, a plasmid construction was obtained allowing the PvEGIII expression in E. coli. Using site-directed mutagenesis, the Asp98Asn mutant of the PvEGIII was obtained. Both the wild type and mutant PvEGIIIs were expressed in E. coli with a yield of up to 1 g/L and then isolated in a highly purified form. The enzyme specific activity against soluble carboxymethylcellulose was not changed after a single amino acid substitution. However, the pH-optimum of activity of the mutant PvEGIII was shifted from pH 4.0 to 5.1, compared to the wild type enzyme. The shift in the enzyme pH-optimum to more neutral pH was also observed on insoluble cellulose, in the process of enzymatic depigmentation of denim fabric. Similar situation featuring the effect of the Asp/Asn residue, located near the Glu catalytic residue, on the enzyme activity pH-profile has previously been described for xylanases of the GH11 family. Thus, the glycoside hydrolases belonging to the GH11 and GH12 families function by a rather similar mechanism of catalysis.

Cloning, purification and comparative characterization of two digestive lysozymes from Musca domestica larvae

cDNA coding for two digestive lysozymes (MdL1 and MdL2) of the Musca domestica housefly was cloned and sequenced. MdL2 is a novel minor lysozyme, whereas MdL1 is the major lysozyme thus far purified from M. domestica midgut. MdL1 and MdL2 were expressed as recombinant proteins in Pichia pastoris, purified and characterized. The lytic activities of MdL1 and MdL2 upon Micrococcus lysodeikticus have an acidic pH optimum (4.8) at low ionic strength (ì = 0.02), which shifts towards an even more acidic value, pH 3.8, at a high ionic strength (ì = 0.2). However, the pH optimum of their activities upon 4-methylumbelliferyl N-acetylchitotrioside (4.9) is not affected by ionic strength. These results suggest that the acidic pH optimum is an intrinsic property of MdL1 and MdL2, whereas pH optimum shifts are an effect of the ionic strength on the negatively charged bacterial wall. MdL2 affinity for bacterial cell wall is lower than that of MdL1. Differences in isoelectric point (pI) indicate that MdL2 (pI = 6.7) is less positively charged than MdL1 (pI = 7.7) at their pH optima, which suggests that electrostatic interactions might be involved in substrate binding. In agreement with that finding, MdL1 and MdL2 affinities for bacterial cell wall decrease as ionic strength increases.