Cytochrome P450 BM-3 Evolved by Random and Saturation Mutagenesis as an Effective Indole-Hydroxylating Catalyst

Cytochrome P450 BM-3 with the mutations A74G, F87V, and L188Q could catalyze indole to produce indigo and indirubin. To further enhance this capability, site-directed and random mutageneses on the monooxygenase domain of P450 BM-3 mutant (A74G/F87V/L188Q; 3X) were performed. The mutant libraries created by error-prone polymerase chain reaction were screened using a colorimetric colony-based method on agar plates followed by a spectroscopic assay involving in absorption of indigo at 670 nm and NADPH at 340 nm in microtiter plate. Three mutants (K434R/3X, E435D/3X, and D168N/A225V/K440N/3X) exhibited higher hydroxylation activity toward indole in comparison to parent enzyme. Moreover, using saturation site-directed mutagenesis at amino acid positions 168, 225, 434, 435, and 440, two P450 BM-3 variants (D168H/3X, E435T/3X) with an up to sixfold increase in catalytic efficiency (k _cat/K _m) were identified, and the mutant D168H/3X acquired higher regioselectivity resulting in more indigo (dimerized 3-hydroxy-indole) compared to parent mutant (93 vs72%).     

Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst

The self-sufficient cytochrome P450 BM-3 enzyme from Bacillus megaterium catalyzes subterminal hydroxylation of saturated long-chain fatty acids and structurally related compounds. Since the primary structure of P450 BM-3 is homologous to that of mammalian P450 type II, it represents an excellent model for this family of enzymes. During studies on the directed evolution of P450 BM-3 into a medium-chain fatty-acid hydroxylase, several mutants, in particular the triple mutant Phe87Val, Leu188Gln, Ala74Gly, were observed to hydroxylate indole, producing indigo and indirubin at a catalytic efficiency of 1365 M(-1)s(-1) (kcat=2.73 s(-1) and Km=2.0 mM). Both products were unequivocally characterized by NMR and MS analysis. Wild-type P450 BM-3 is incapable to hydroxylate indole. These results demonstrate that an enzyme can be engineered to catalyze the transformation of substrates with structures widely divergent from those of its native substrate.     

Directed Evolution of a Thermophilic β-glucosidase for Cellulosic Bioethanol Production

Characteristics that would make enzymes more desirable for industrial applications can be improved using directed evolution. We developed a directed evolution technique called random drift mutagenesis (RNDM). Mutant populations are screened and all functional mutants are collected and put forward into the next round of mutagenesis and screening. The goal of this technique is to evolve enzymes by rapidly accumulating mutations and exploring a greater sequence space by providing minimal selection pressure and high-throughput screening. The target enzyme was a β-glucosidase isolated from the thermophilic bacterium, Caldicellulosiruptor saccharolyticus that cleaves cellobiose resulting from endoglucanase hydrolysis of cellulose. Our screening method was fluorescence-activated cell sorting (FACS), an attractive method for assaying mutant enzyme libraries because individual cells can be screened, sorted into distinct populations and collected very rapidly. However, FACS screening poses several challenges, in particular, maintaining the link between genotype and phenotype because most enzyme substrates do not remain associated with the cells. We employed a technique where whole cells were encapsulated in cell-like structures along with the enzyme substrate. We used RNDM, in combination with whole cell encapsulation, to create and screen mutant β-glucosidase libraries. A mutant was isolated that, compared to the wild type, had higher specific and catalytic efficiencies (k _cat/K _M) with p-nitrophenol-glucopyranoside and -galactopyranoside, an increased catalytic turnover rate (k _cat) with cellobiose, an improvement in catalytic efficiency with lactose and reduced inhibition (K _i) with galactose and lactose. This mutant had three amino acid substitutions and one was located near the active site.     

Cloning and expression of NDM-1 metallo-β-lactamase gene and study of the catalytic properties of the recombinant enzyme

A gene-expression system has been designed to express the NDM-1 metallo-β-lactamase gene in E. coli cells. This system enables the synthesis of the recombinant protein in a soluble and active form. A method for the isolation and purification of the recombinant enzyme has been developed. The yield of the homogeneous protein preparation was 10–15 mg per liter of E. coli culture medium. The catalytic parameters of the recombinant NDM-1 β-lactamase were measured for ampicillin (K_m = 185 μM and k_cat = 585 s^–1) and meropenem (K_m = 85 μM and k_cat = 160 s^–1). These values correlate well with the literature data. The catalytic parameters for the chromogenic CENTA substrate (K_m = 14 μM and k_cat = 290 s^–1) were obtained for the first time.     

Water Miscible Mono Alcohols’ Effect on the Proteolytic Performance of Bacillus clausii Serine Alkaline Protease

In this study, our investigations showed that the increasing concentrations of all examined mono alcohols caused a decrease in the V _m, k _cat and k _cat/K _m values of Bacillus clausii GMBE 42 serine alkaline protease for casein hydrolysis. However, the K _m value of the enzyme remained almost the same, which was an indicator of non-competitive inhibition. Whereas inhibition by methanol was partial non-competitive, inhibition by the rest of the alcohols tested was simple non-competitive. The inhibition constants (K _I) were in the range of 1.32–3.10 M, and the order of the inhibitory effect was 1-propanol>2-propanol>methanol>ethanol. The ΔG ^≠ and ΔG ^≠ _E???T values of the enzyme increased at increasing concentrations of all alcohols examined, but the ΔG ^≠ _ES value of the enzyme remained almost the same. The constant K _m and ΔG ^≠ _ES values in the presence and absence of mono alcohols indicated the existence of different binding sites for mono alcohols and casein on enzyme the molecule. The k _cat of the enzyme decreased linearly by increasing log P and decreasing dielectric constant (D) values, but the ΔG ^≠ and ΔG ^≠ _E???T values of the enzyme increased by increasing log P and decreasing D values of the reaction medium containing mono alcohols.     

Activity Enhancement of Candida antarctica Lipase B by Flexibility Modulation in Helix Region Surrounding the Active Site

The activity of Candida antarctica lipase B was improved by mutation of the area surrounding the active site. We changed the edges of four helices surrounding the active site to flexible amino acids. Two mutants, V139E and I255E, obtained as a result of Pichia pastoris expression, showed enhanced specific activity of 9.9 and 8.1 U/mg while that of wild type was 2.3 U/mg for p-nitrophenyl caprylate hydrolysis. It was nearly 5.4-fold and 3.5-fold, respectively. The stability of both mutants on organic solvent was slightly decreased but almost similar with that of wild type. In the kinetic assay, k _cat values were shown as dominant factor for the enhancement of catalytic efficiency, k _cat/K _m, since it was 4.1-fold and 3.8-fold, respectively.     

Enhanced Activity and Substrate Specificity by Site-Directed Mutagenesis for the P450 119 Peroxygenase Catalyzed Sulfoxidation of Thioanisole

P450 119 peroxygenase was found to catalyze the sulfoxidation of thioanisole and the sulfonation of sulfoxide in the presence of tert-butyl hydroperoxide (TBHP) for the first time with turnover rates of 1549 min⁻¹ and 196 min⁻¹ respectively. Several mutants were designed to improve the peroxygenation activity and thioanisole specificity by site-directed mutagenesis. The F153G/T213G mutant gave an increase of sulfoxide yield and a decrease of sulfone yield. Moreover the S148P/I161T/K199E/T214V mutant and the K199E mutant with acidic Glu residue contributed to improving the product ratio of sulfoxide to sulfone. Addition of short-alkyl-chain organic acids to the P450 119 peroxygenase-catalyzed sulfur oxidation of thioanisole was investigated. Octanoic acid was found to induce a preferred sulfoxidation of thioanisole catalyzed by the F153G/T213G mutant to give approximately 2.4-fold increase in turnover rate with a k_cat value of 3687 min⁻¹ relative to that of the wild-type, and by the F153G mutant to give the R-sulfoxide up to 30 % ee. The experimental control and the proposed mechanism for the P450 119 peroxygenase-catalyzed sulfoxidation of thioanisole in the presence of octanoic acid suggested that octanoic acid could partially occupy the substrate pocket; meanwhile the F153G mutation could enhance the substrate specificity, which could lead to efficiently regulate the spatial orientation of thioanisole and facilitate the formation of Compound I. This is the most effective catalytic system for the P450 119 peroxygenase-catalyzed sulfoxidation of thioanisole.     

Tuning the regio- and stereoselectivity of C-H activation in n-octanes by cytochrome P450 BM-3 with fluorine substituents: evidence for interactions between a C-F bond and aromatic π systems

We employed the water-soluble cytochrome P450 BM-3 to study the activity and regiospecificity of oxidation of fluorinated n-octanes. Three mutations, A74G, F87V, and L188Q, were introduced into P450 BM-3 to allow the system to undergo n-octane oxidation. In addition, the alanine at residue 328 was replaced with a phenylalanine to introduce an aromatic residue into the hydrophobic pocket to examine whether or not van der Waals interactions between a C-F substituent in the substrate and the polarizable π system of the phenylalanine may be used to steer the positioning of the substrate within the active-site pocket of the enzyme and control the regioselectivity and stereoselectivity of hydroxylation. Interestingly, not only was the regioselectivity controlled when the fluorine substituent was judiciously positioned in the substrate, but the electron input into the iron-heme group became tightly coupled to the formation of product, essentially without abortive side reactions. Remarkable enhancement of the coupling efficiency between electron input and product formation was observed for a range of fluorinated octanes in the enzyme even without the A328F mutation, presumably because of interactions of the C-F substituent with the π system of the porphyrin macrocycle within the active-site pocket. Evidently, tightening the protein domain containing the heme pocket tunes the distribution of accessible enzyme conformations and the associated protein dynamics that activate the iron porphyrin for substrate hydroxylation to allow the reactions mediated by the high-valent Fe(IV)=O to become kinetically more commensurate with electron transfer from the flavin adenine dinucleotide (FAD)/flavin mononucleotide (FMN) reductase. These observations lend compelling evidence to support significant van der Waals interactions between the CF(2) group and aromatic π systems within the heme pocket when the fluorinated octane substrate is bound.     

Single-Point Mutant Inverts the Stereoselectivity of a Carbonyl Reductase toward β-Ketoesters with Enhanced Activity

Enzyme stereoselectivity control is still a major challenge. To gain insight into the molecular basis of enzyme stereo-recognition and expand the source of antiPrelog carbonyl reductase toward β-ketoesters, rational enzyme design aiming at stereoselectivity inversion was performed. The designed variant Q139G switched the enzyme stereoselectivity toward β-ketoesters from Prelog to antiPrelog, providing corresponding alcohols in high enantiomeric purity (89.1–99.1 % ee). More importantly, the well-known trade-off between stereoselectivity and activity was not found. Q139G exhibited higher catalytic activity than the wildtype enzyme, the enhancement of the catalytic efficiency (k_cat/K_m) varied from 1.1- to 27.1-fold. Interestingly, the mutant Q139G did not lead to reversed stereoselectivity toward aromatic ketones. Analysis of enzyme–substrate complexes showed that the structural flexibility of β-ketoesters and a newly formed cave together facilitated the formation of the antiPrelog-preferred conformation. In contrast, the relatively large and rigid structure of the aromatic ketones prevents them from forming the antiPrelog-preferred conformation.     

Enzymatic Activation of the Emerging Drug Resveratrol

The plant originated stilbene “resveratrol” (3,4′,5-trans-trihydroxystilbene) is well known for its diverse health benefits including anti-tumor, anti-inflammatory, anti-microbial, and anti-oxidant properties. Besides a significant amount of reports on different aspects of its application as prodrug in the last 50 years, still, a strategy leading to the production of the active drug is missing. The aim of this work was to evaluate the enzymatic activation of prodrug resveratrol to the effective drug piceatannol, without engaging expensive cofactors. Five different heme proteins were analyzed for the transformation of resveratrol. Kinetic parameters of resveratrol transformation and analysis of the transformed products were conducted through HPLC and GC-MS. Effect of pH and organic solvent on the transformation process had also been evaluated. Among all tested heme proteins, only a variant of cytochrome P450_BM3 from Bacillus megaterium (CYP_BM3F87A) was found suitable for piceatannol production. The most suitable pH for the reaction conditions was 8.5, while organic solvents did not show any effect on transformation. For resveratrol transformation, the turnover rate (k _cat) was 21.7 (± 0.6) min^?1, the affinity constant (K _M) showed a value of 55.7 (± 16.7) μM for a catalytic efficiency (k _cat/K _M) of 389 min^?1 mM^?1. GC-MS analysis showed that the only product from resveratrol transformation by cytochrome P450_BM3 is the biologically active piceatannol. The enzymatic transformation of resveratrol, an emerging compound with medical interest, to active product piceatannol by a variant of cytochrome P450_BM3 in the absence of expensive NADPH cofactor is demonstrated. This enzymatic process is economically attractive and can be scaled up to cover the increasing medical demand for piceatannol.     

Kinetic Properties of Two Rhizopus Exo-polygalacturonase Enzymes Hydrolyzing Galacturonic Acid Oligomers Using Isothermal Titration Calorimetry

The kinetic characteristics of two Rhizopus oryzae exo-polygalacturonases acting on galacturonic acid oligomers (GalpA) were determined using isothermal titration calorimetry (ITC). RPG15 hydrolyzing (GalpA)₂ demonstrated a K _m of 55 μM and k _cat of 10.3 s^?1 while RPG16 was shown to have greater affinity for (GalpA)₂ with a K _m of 16 μM, but lesser catalytic activity with a k _cat of 3.9 s^?1. Both enzymes were inhibited by the product, galacturonic acid, with _app K _i values of 886 and 501 μM for RPG15 and RPG16, respectively. RPG15 exhibited greater affinity for (GalpA)₃ with a K _m of 9.2 μM and a similar k _cat at 10.7 s^?1 relative to (GalpA)₂. Catalytic constants for RPG16 hydrolyzing (GalpA)₃ could not be determined; however, single-injection ITC assays suggest a distinct preference and catalytic rate for (GalpA)₃ relative to (GalpA)₂. Thermodynamic parameters of a series of galacturonic acid oligomers binding to RPG15 were determined and exhibited some distinct differences from RPG16 binding thermodynamics, providing potential clues to the differing kinetic characteristics of the two exo-polygalacturonase enzymes.     

β-d-Xylosidase from Selenomonas ruminantium: Thermodynamics of Enzyme-Catalyzed and Noncatalyzed Reactions

β-d-Xylosidase/α-l-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme known for catalyzing hydrolysis of 1,4-β-d-xylooligosaccharides to d-xylose. Temperature dependence for hydrolysis of 4-nitrophenyl-β-d-xylopyranoside (4NPX), 4-nitrophenyl-α-l-arabinofuranoside (4NPA), and 1,4-β-d-xylobiose (X2) was determined on and off (k _non) the enzyme at pH 5.3, which lies in the pH-independent region for k _cat and k _non. Rate enhancements (k _cat/k _non) for 4NPX, 4NPA, and X2 are 4.3?×?10¹¹, 2.4?×?10⁹, and 3.7?×?10¹², respectively, at 25 °C and increase with decreasing temperature. Relative parameters k _cat ^4NPX/k _cat ^4NPA, k _cat ^4NPX/k _cat ^X2, and (k _cat/K _m)^4NPX/(k _cat/K _m)^X2 increase and (k _cat/K _m)^4NPX/(k _cat/K _m)^4NPA, (1/K _m)^4NPX/(1/K _m)^4NPA, and (1/K _m)^4NPX/(1/K _m)^X2 decrease with increasing temperature.     

Discovery of Selective Small‐Molecule Activators of a Bacterial Glycoside Hydrolase

Fragment‐based approaches are used routinely to discover enzyme inhibitors as cellular tools and potential therapeutic agents. There have been few reports, however, of the discovery of small‐molecule enzyme activators. Herein, we describe the discovery and characterization of small‐molecule activators of a glycoside hydrolase (a bacterial O‐GlcNAc hydrolase). A ligand‐observed NMR screen of a library of commercially available fragments identified an enzyme activator which yielded an approximate 90 % increase in k_cat/K_M values (k_cat=catalytic rate constant; K_M=Michaelis constant). This compound binds to the enzyme in close proximity to the catalytic center. Evolution of the initial hits led to improved compounds that behave as nonessential activators effecting both K_M and V_max values (V_max=maximum rate of reaction). The compounds appear to stabilize an active “closed” form of the enzyme. Such activators could offer an orthogonal alternative to enzyme inhibitors for perturbation of enzyme activity in vivo, and could also be used for glycoside hydrolase activation in many industrial processes.     

Accelerating Enzymatic Catalysis Using Vortex Fluidics

Enzymes catalyze chemical transformations with outstanding stereo‐ and regio‐specificities, but many enzymes are limited by their long reaction times. A general method to accelerate enzymes using pressure waves contained within thin films is described. Each enzyme responds best to specific frequencies of pressure waves, and an acceleration landscape for each protein is reported. A vortex fluidic device introduces pressure waves that drive increased rate constants (k_cat) and enzymatic efficiency (k_cat/K_m). Four enzymes displayed an average seven‐fold acceleration, with deoxyribose‐5‐phosphate aldolase (DERA) achieving an average 15‐fold enhancement using this approach. In solving a common problem in enzyme catalysis, a powerful, generalizable tool for enzyme acceleration has been uncovered. This research provides new insights into previously uncontrolled factors affecting enzyme function.     

Improving the activity of immobilized subtilisin by site-directed attachment through a genetically engineered affinity tag

An octapeptide affinity tag, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (termed FLAG), was genetically fused to the C-terminus of subtilisin BPN′ (SBT) from Bacillus amyloliquefaciens. The fusion protein SBT-FLAG was immobilized to nonporous polystyrene and silica beads both in a site-directed and a random fashion. Site-directed immobilization was achieved by employing the interaction between protein A and a monoclonal antibody specific for the FLAG peptide, while random immobilization was obtained by using glutaraldehyde as a cross-linking reagent. The activity of the immobilized enzymes was compared. It was found that the site-directed subtilisin had higher catalytic efficiency, k_cat/K_M, which was more than 7-fold of that of the randomly immobilized enzyme. It was also noted that the site-directly immobilized enzyme had superior storage stability over the homogeneous enzyme.     

Purification and Characterization of a Glycoside Hydrolase Family 43 β-xylosidase from Geobacillus thermoleovorans IT-08

The gene encoding a glycoside hydrolase family 43 β-xylosidase (GbtXyl43A) from the thermophilic bacterium Geobacillus thermoleovorans strain IT-08 was synthesized and cloned with a C-terminal His-tag into a pET29b expression vector. The recombinant gene product termed GbtXyl43A was expressed in Escherichia coli and purified to apparent homogeneity. Michaelis–Menten kinetic parameters were obtained for the artificial substrates p-nitrophenyl-β-d-xylopyranose (4NPX) and p-nitrophenyl-α-l-arabinofuranose (4NPA), and it was found that the ratio k _cat/K _m 4NPA/k _cat/K _m 4NPX was ～7, indicting greater catalytic efficiency for 4NP hydrolysis from the arabinofuranose aglycon moiety. Substrate inhibition was observed for the substrates 4-methylumbelliferyl xylopyranoside (muX) and the arabinofuranoside cogener (muA), and the ratio k _cat/K _m muA/k _cat/K _m muX was ～5. The enzyme was competitively inhibited by monosaccharides, with an arabinose K _i of 6.8?±?0.62 mM and xylose K _i of 76?±?8.5 mM. The pH maxima was 5.0, and the enzyme was not thermally stable above 54 °C, with a t _1/2 of 35 min at 57.5 °C. GbtXyl43A showed a broad substrate specificity for hydrolysis of xylooligosaccharides up to the highest degree of polymerization tested (xylopentaose), and also released xylose from birch and beechwood arabinoxylan.     

The differential modulation of USP activity by internal regulatory domains, interactors and eight ubiquitin chain types

Ubiquitin-specific proteases (USPs) are papain-like isopeptidases with variable inter- and intramolecular regulatory domains. To understand the effect of these domains on USP activity, we have analyzed the enzyme kinetics of 12 USPs in the presence and absence of modulators using synthetic reagents. This revealed variations of several orders of magnitude in both the catalytic turnover (k_cat) and ubiquitin (Ub) binding (K_M) between USPs. Further activity modulation by intramolecular domains affects both the k_cat and K_M, whereas the intermolecular activators UAF1 and GMPS mainly increase the k_cat. Also, we provide the first comprehensive analysis comparing Ub chain preference. USPs can hydrolyze all linkages and show modest Ub-chain preferences, although some show a lack of activity toward linear di-Ub. This comprehensive kinetic analysis highlights the variability within the USP family.     

Molecular modeling of cytochrome P450 3A4

The three-dimensional structure of human cytochrome P450 3A4 was modeled based on crystallographic coordinates of four bacterial P450s: P450 BM-3, P450cam, P450terp, and P450eryF. The P450 3A4 sequence was aligned to those of the known proteins using a structure-based alignment of P450 BM-3, P450cam, P450terp, and P450eryF. The coordinates of the model were then calculated using a consensus strategy, and the final structure was optimized in the presence of water. The P450 3A4 model resembles P450 BM-3 the most, but the B helix is similar to that of P450eryF, which leads to an enlarged active site when compared with P450 BM-3, P450cam, and P450terp. The 3A4 residues equivalent to known substrate contact residues of the bacterial proteins and key residues of rat P450 2B1 are located in the active site or the substrate access channel. Docking of progesterone into the P450 3A4 model demonstrated that the substrate bound in a 6-orientation can interact with a number of active site residues, such as 114, 119, 301, 304, 305, 309, 370, 373, and 479, through hydrophobic interactions. The active site of the enzyme can also accommodate erythromycin, which, in addition to the residues listed for progesterone, also contacts residues 101, 104, 105, 214, 215, 217, 218, 374, and 478. The majority of 3A4 residues which interact with progesterone and/or erythromycin possess their equivalents in key residues of P450 2B enzymes, except for residues 297, 480 and 482, which do not contact either substrate in P450 3A4. The results from docking of progesterone and erythromycin into the enzyme model make it possible to pinpoint residues which may be important for 3A4 function and to target them for site-directed mutagenesis.     

Functional contribution of coenzyme specificity-determining sites of 7α-hydroxysteroid dehydrogenase from Clostridium absonum

Studies of the molecular determinants of coenzyme specificity help to reveal the structure-function relationship of enzymes, especially with regards to coenzyme specificity-determining sites (CSDSs) that usually mediate complex interactions. NADP(H)-dependent 7α-hydroxysteroid dehydrogenase from Clostridium absonum (CA 7α-HSDH), a member of the short-chain dehydrogenase/reductase superfamily (SDRs), possesses positively charged CSDSs that mainly contain T15, R16, R38, and R194, forming complicated polar interactions with the adenosine ribose C2 phosphate group of NADP(H). The R38 residue is crucial for coenzyme anchoring, but the influence of the other residues on coenzyme utilization is still not clear. Hence, we performed alanine scanning mutagenesis and molecular dynamic (MD) simulations. The results suggest that the natural CSDSs have the greatest NADP(H)-binding affinity, but not the best activity (k_cat) toward NADP⁺. Compared with the wild type and other mutants, the mutant R194A showed the highest catalytic efficiency (k_cat/K_m), which was more than three-times that of the wild type. MD simulation and kinetics analysis suggested that the importance of the CSDSs of CA 7α-HSDH should be in accordance with the following order R38 > T15 > R16 > R194, and S39 may have a supporting role in NADP(H) anchoring for mutants R16A/T194A and T15A/R16A/T194A.     

Purification and Analytical Application of <Emphasis Type="Italic">Vigna mungo</Emphasis> Chitinase for Determination of Total Fungal Load of Stored Cereals

Chitinases are glycosyl hydrolases that catalyze the hydrolysis of β-(1,4)-glycosidic bonds in chitin, the major structural polysaccharide presented in the cuticle and gut peritrophic matrix of insects. Two aspartate residues (D143, D145) and one tryptophan (W146) in the Lymantria dispar chitinase are highly conserved residues observed within the second conserved motif of the family 18 chitinase catalytic region. In this study, a chitinase cDNA, LdCht5, was cloned from L. dispar, and the roles of the three residues were investigated using site-directed mutagenesis and substituting them with three other amino acids. Seven mutant proteins, D143E, D145E, W146G, D143E/D145E, D143E/W146G, D145E/W146G, and D143E/D145E/W146G, as well as the wild-type enzyme, were produced using the baculovirus-insect cell line expression system. The enzymatic and kinetic properties of these mutant enzymes were measured using the oligosaccharide substrate MU-(GlcNAc)₃. Among the seven mutants, the D145E, D143E/D145E, and D145E/W146G mutations kept some extant catalytic activity toward MU-(GlcNAc)₃, while the D143E, W146G, D143E/W146G, and D143E/D145E/W146G mutant enzymes were inactivated. Compared with the mutant enzymes, the wild-type enzyme had higher values of k _cat and k _cat / K _m . A study of the multiple point mutations in the second conserved catalytic region would help to elucidate the role of the critical residues and their relationships.