Residue Asn277 Affects the Stability and Substrate Specificity of the SMG1 Lipase from Malassezia globosa

Thermostability and substrate specificity are important characteristics of enzymes for industrial application, which can be improved by protein engineering. SMG1 lipase from Malassezia globosa is a mono- and diacylglycerol lipase (MDL) that shows activity toward mono- and diacylglycerols, but no activity toward triacylglycerols. SMG1 lipase is considered a potential biocatalyst applied in oil/fat modification and its crystal structure revealed that an interesting residue-Asn277 may contribute to stabilize loop 273–278 and the 3₁₀4 helix which are important to enzyme characterization. In this study, to explore its role in affecting the stability and catalytic activity, mutagenesis of N277 with Asp (D), Val (V), Leu (L) and Phe (F) was conducted. Circular dichroism (CD) spectral analysis and half-life measurement showed that the N277D mutant has better thermostability. The melting temperature and half-life of the N277D mutant were 56.6 °C and 187 min, respectively, while that was 54.6 °C and 121 min for SMG1 wild type (WT). Biochemical characterization of SMG1 mutants were carried out to test whether catalytic properties were affected by mutagenesis. N277D had similar enzymatic properties as SMG1 WT, but N277F showed a different substrate selectivity profile as compared to other SMG1 mutants. Analysis of the SMG1 3D model suggested that N277D formed a salt bridge via its negative charged carboxyl group with a positively charged guanidino group of R227, which might contribute to confer N277D higher temperature stability. These findings not only provide some clues to understand the molecular basis of the lipase structure/function relationship but also lay the framework for engineering suitable MDL lipases for industrial applications.     

Roles of catalytic residues in {alpha}-amylases as evidenced by the structures of the product-complexed mutants of a maltotetraose-forming amylase

The crystal structures of the four product-complexed singlemutants of the catalytic residues of Pseudomonas stutzeri maltotetraose-forming-amylase, E219G, D193N, D193G and D294N, have been determined.Possible roles of the catalytic residues Glu219, Asp193 andAsp294 have been discussed by comparing the structures amongthe previously determined complexed mutant E219Q and the presentmutant enzymes. The results suggested that Asp193 predominantlyworks as the base catalyst (nucleophile), whose side chain atomlies in close proximity to the C1-atom of Glc4, being involvedin the intermediate formation in the hydrolysis reaction. WhileAsp294 works for tightly binding the substrate to give a twistedand a deformed conformation of the glucose ring at position–1 (Glc4). The hydrogen bond between the side chain atomof Glu219 and the O1-atom of Glc4, that implies the possibilityof interaction via hydrogen, consistently present throughoutthese analyses, supports the generally accepted role of thisresidue as the acid catalyst (proton donor).     

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

In order to understand better the structural and functionalrelations between protein kinase CK2 catalytic subunit, thetriphosphate moiety of ATP, the catalytic metal and the peptidicsubstrate, we built a structural model of Yarrowia lipolyticaprotein kinase CK2 catalytic subunit using the recently solvedthree-dimensional structure of the maize enzyme and the structureof cAMP-dependent protein kinase peptidic inhibitor (1CDK) astemplates. The overall structure of the catalytic subunit isclose to the structure solved by Niefind et al. It comprisestwo lobes, which move relative to each other. The peptide usedas substrate is tightly bound to the enzyme, at specific locations.Molecular dynamic calculations in combination with the studyof the structural model led us to identify amino acid residuesclose to the triphosphate moiety of ATP and a residue sufficientlyfar from the peptide that could be mutated so as to modify thespecificity of the enzyme. Site-directed mutagenesis was usedto replace by charged residues both glycine-48, a residue locatedwithin the glycine-rich loop, involved in binding of ATP phosphatemoiety, and glycine-177, a residue close to the active site.Kinetic properties of purified wild-type and mutated subunitswere studied with respect to ATP, MgCl₂ and protein kinase CK2specific peptide substrates. The catalytic efficiency of theG48D mutant increased by factors of 4 for ATP and 17.5 for theRRRADDSDDDDD peptide. The mutant G48K had a low activity withATP and no detectable activity with peptide substrates and wasalso inhibited by magnesium. An increased velocity of ADP releaseby G48D and the building of an electrostatic barrier betweenATP and the peptidic substrate in G48K could explain these results.The kinetic properties of the mutant G177K with ATP were notaffected, but the catalytic efficiency for the RRRADDSDDDDDsubstrate increased sixfold. Lysine 177 could interact withthe lysine-rich cluster involved in the specificity of proteinkinase CK2 towards acidic substrate, thereby increasing itsactivity.     

The role of asparagine-32 in forming the receptor-binding epitope of human epidermal growth factor

The highly conserved asparagine residue at position 32 (Asn32)in the 'hinge' region of epidermal growth factor (EGF) separatesthe N- and C-terminal structural motifs of the EGF moleculeand is therefore an appropriate target for structure-functionstudies. Analogs of human EGF (hEGF) were generated in whichAsn32 was substituted with aspartate, glycine, isoleucine, lysine,proUne and tryptophan. The relative affinity of the EGF receptorfor mutant hEGF analogs was determined by radioreceptor competitionassay. A wide range of receptor affinities was observed dependingon the amino acid substitution. N32K and N32W hEGF analogs hadrelatively high receptor affinity, while the N32G and N32D analogsshowed decreased affinity, 35% and 25% respectively, relativeto wild type hEGF. However, no binding of the N32P analog wasdetected by radioreceptor competition assay. The N32P mutantdisplayed an NMR spectrum significantly different from thatof native wild type hEGF, indicating gross structural perturbation.In contrast, the N32K and N32D analogs exhibited spectra similarto that of native wild type hEGF. Genetically combining theN32D hEGF with an hEGF species having either the mutation L26Ghi the N-terminal region or L47A in the C-terminal region, generateddouble-mutant hEGF species whkh had relative affinities essentiallyequal to the product of the relative affinities of the parenthEGF mutants, indicating functionally independent changes inUgand-receptor interaction. These studies indicate the requirementfor H-bond donor functionality in the side chain of residuenumber 32 in forming a fully competent receptor-binding epitope.     

Revisiting the mechanism of the triosephosphate isomerase reaction: the role of the fully conserved glutamic acid 97 residue

An analysis of 503 available triosephosphate isomerase sequences revealed nine fully conserved residues. Of these, four residues—K12, H95, E97 and E165—are capable of proton transfer and are all arrayed around the dihydroxyacetone phosphate substrate in the three‐dimensional structure. Specific roles have been assigned to the residues K12, H95 and E165, but the nature of the involvement of E97 has not been established. Kinetic and structural characterization is reported for the E97Q and E97D mutants of Plasmodium falciparum triosephosphate isomerase (Pf TIM). A 4000‐fold reduction in k_cat is observed for E97Q, whereas the E97D mutant shows a 100‐fold reduction. The control mutant, E165A, which lacks the key catalytic base, shows an approximately 9000‐fold drop in activity. The integrity of the overall fold and stability of the dimeric structure have been demonstrated by biophysical studies. Crystal structures of E97Q and E97D mutants have been determined at 2.0 Å resolution. In the case of the isosteric replacement of glutamic acid by glutamine in the E97Q mutant a large conformational change for the critical K12 side chain is observed, corresponding to a trans‐to‐gauche transition about the Cγ? Cδ (χ³) bond. In the E97D mutant, the K12 side chain maintains the wild‐type orientation, but the hydrogen bond between K12 and D97 is lost. The results are interpreted as a direct role for E97 in the catalytic proton transfer cycle. The proposed mechanism eliminates the need to invoke the formation of the energetically unfavourable imidazolate anion at H95, a key feature of the classical mechanism.     

Engineering the active center of the 6-phospho-{beta}-galactosidase from Lactococcus lactis

Several amino acids in the active center of the 6-phospho-ß-galactosidasefrom Lactococcus lactis were replaced by the corresponding residuesin homologous enzymes of glycosidase family 1 with differentspecificities. Three mutants, W429A, K435V/Y437F and S428D/K435V/Y437F, were constructed. W429A was found to have an improvedspecificity for glucosides compared with the wild-type, consistentwith the theory that the amino acid at this position is relevantfor the distinction between galactosides and glucosides. Thek_cat/K_m for o-nitrophenyl-ß-D-glucose-6-phosphate is 8-foldhigher than for o-nitrophenyl-ß-D-galactose-6-phosphatewhich is the preferred substrate of the wild-type enzyme. Thissuggests that new hydrogen bonds are formed in the mutant betweenthe active site residues, presumably Gln19 or Trp421 and theC-4 hydroxyl group. The two other mutants with the exchangesin the phosphate-binding loop were tested for their abilityto bind phosphorylated substrates. The triple mutant is inactive.The double mutant has a dramatically decreased ability to bindo-nitrophenyl-ß-D-galactose-6-phosphate whereas the interactionwith o-nitrophenyl-ß-D-galactose is barely altered. Thisresult shows that the 6-phospho-ß-galactosidase and therelated cyanogenic ß-glucosidase from Trifolium repenshave different recognition mechanisms for substrates althoughthe structures of the active sites are highly conserved.     

In silico mutations and molecular dynamics studies on a winged bean chymotrypsin inhibitor protein

Winged bean chymotrypsin inhibitor (WCI) has an intruding residueAsn14 that plays a crucial role in stabilizing the reactivesite loop conformation. This residue is found to be conservedin the Kunitz (STI) family of serine protease inhibitors. Tounderstand the contribution of this scaffolding residue on thestability of the reactive site loop, it was mutated in silicoto Gly, Ala, Ser, Thr, Leu and Val and molecular dynamics (MD)simulations were carried out on the mutants. The results ofMD simulations reveal the conformational variability and rangeof motions possible for the reactive site loop of differentmutants. The N-terminus side of the scissile bond, which isclose to a ß-barrel, is conformationally less variable,while the C-terminus side, which is relatively far from anysuch secondary structural element, is more variable and needsstability through hydrogen-bonding interactions. The simulatedstructures of WCI and the mutants were docked in the peptide-bindinggroove of the cognate enzyme chymotrypsin and the ability toform standard hydrogen-bonding interactions at P3, P1 and P2'residues were compared. The results of the MD simulations coupledwith docking studies indicate that hydrophobic residues likeLeu and Val at the 14th position are disruptive for the integrityof the reactive site loop, whereas a residue like Thr, whichcan stabilize the C-terminus side of the scissile bond, canbe predicted at this position. However, the size and chargeof the Asn residue made it most suitable for the best maintenanceof the integrity of the reactive site loop, explaining its conservednature in the family. Received October 17, 2002; revised June 6, 2003; accepted June 19, 2003.     

Triple point mutation Asp10 -> His, Asn101 -> Asp, Argl48 -> Ser in T4 phage lysozyme leads to the molten globule

The triple amino acid replacement (Asp10 His, Asn101 Asp,Arg148 Ser) in T4 phage lysozyme was carried out by site-directedmutagenesis. At acid pH (2.7) the mutant is in a confonnationalstate with the properties of the molten globule: (i) the mutantprotein molecule is essentially compact; (ii) its CD spectrumin the near UV region is drastically reduced in intensity ascompared with the wild type protein spectrum; (iii) the CD spectrumin the far UV region indicates the presence of pronounced secondarystructure in the mutant; (iv) unlike the wild type protein themutant protein can bind the hydrophobic fluorescent probe, ANS.     

Proposal for new catalytic roles for two invariant residues in Escherichia coli ribonuclease HI

Three mutants of Escherichia coli ribonuclease HI, in whichan invariant acidic residue Asp134 was replaced, were crystallized,and their three-dimensional structures were determined by X-raycrystallography. The D134A mutant is completely inactive, whereasthe other two mutants, D134H and D134N, retain 59 and 90% activitiesrelative to the wild-type, respectively. The overall structuresof these three mutant proteins are identical with that of thewild-type enzyme, except for local conformational changes ofthe flexible loops. The ribonuclease H family has a common activesite, which is composed of four invariant acidic residues (Asp10,G1u48, Asp70 and Asp134 in E.coli ribonuclease HI), and theirrelative positions in the mutants, even including the side-chainatoms, are almost the same as those in the wild-type. The positionsof the -polar atoms at residue 134 in the wild-type, as wellas D134H and D134N, coincide well with each other. They arelocated near the imidazole side chain of His124, which is assumedto participate in the catalytic reaction, in addition to thefour invariant acidic residues. Combined with the pH profilesof the enzymatic activities of the two other mutants, H124Aand H124A/D134N, the crystallographic results allow us to proposea new catalytic mechanism of ribonuclease H, which includesthe roles for Asp134 and His124.     

Molecular recognition at the active site of subtilisin BPN': crystallographic studies using genetically engineered proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor)

Unlike trypsin-like serine proteases having only one conspicuousbinding pocket in the active site, subtilisin BPN' has two suchpockets, the S1 and S4 pockets, which accommodate the P1 andP4 residues of ligands (after Schechter and Berger notation)respectively. Using computer graphics, the geometrical natureof the two pockets was carefully examined and strategies forsite-directed mutagenesis studies were set up against a proteinSSI (Streptomyces subtilisin inhibitor), which is a strong proteinaceousinhibitor (or a substrate analogue) of subtilisin BPN'. It wasdecided to convert the P1 residue, methionine 73, into lysine(M73K) with or without additional conversion of the P4 residue,methionine 70, into glycine (M70G). The crystal structures ofthe two complexes of subtilisin BPN', one with the single mutantSSI (M73K) and the other with the double mutant SSI (M73K, M70G)were solved showing that (i) small ‘electrostatic induced-fitmovement’ occurs in the S1 pocket upon introducing theterminal plus charge of the lysine side chain, and (ii) large‘mechanical induced-fit movement’ occurs in theS4 pocket upon reducing the size of the P4 side chain from methionineto glycine. In both (i) and (ii), the induced-fit movement occurredin a concerted fashion involving both the enzyme and ‘substrate’amino acid residues. The term ‘substrate-assisted stabilization’was coined to stress the cooperative nature of the induced-fitmovements.     

Essential dynamics of lipase binding sites: the effect of inhibitors of different chain length

The biochemical activity of enzymes, such as lipases, is often associated with structural changes in the enzyme resulting in selective and stereospecific reactions with the substrate. To investigate the effect of a substrate and its chain length on the dynamics of the enzyme, we have performed molecular dynamics simulations of the native Rhizomucor miehei lipase (Rml) and lipase-dialkylphosophate complexes, where the length of the alkyl chain ranges from two to 10 carbon atoms. Simulations were performed in water and trajectories of 400 ps were used to analyse the essential motions in these systems. Our results indicate that the internal motions of the Rml and Rml complexes occur in a subspace of only a few degrees of freedom. A high flexibility is observed in solvent-exposed segments, which connect beta-sheets and helices. In particular, loop regions Gly35-Lys50 and Thr57-Asn63 fluctuate extensively in the native enzyme. Upon activation and binding of the inhibitor, involving the displacement of the active site loop, these motions are considerably suppressed. With increasing chain length of the inhibitor, the fluctuations in the essential subspace increase, levelling off at a chain length of 10, which corresponds to the size of the active-site groove.      

Synthesis, biological activity, and ADME properties of novel S-DABOs/N-DABOs as HIV reverse transcriptase inhibitors

Previous studies aimed at exploring the SAR of C2-functionalized S-DABOs demonstrated that the substituent at this position plays a key role in the inhibition of both wild-type RT and drug-resistant enzymes, particularly the K103N mutant form. The introduction of a cyclopropyl group led us to the discovery of a potent inhibitor with picomolar activity against wild-type RT and nanomolar activity against many key mutant forms such as K103N. Despite its excellent antiviral profile, this compound suffers from a suboptimal ADME profile typical of many S-DABO analogues, but it could, however, represent a promising candidate as an anti-HIV microbicide. In the present work, a new series of S-DABO/N-DABO derivatives were synthesized to obtain additional SAR information on the C2-position and in particular to improve ADME properties while maintaining a good activity profile against HIV-1 RT. In vitro ADME properties (PAMPA permeation, water solubility, and metabolic stability) were also experimentally evaluated for the most interesting compounds to obtain a reliable indication of their plasma levels after oral administration.     

Preferential labeling of brain cholesterol by {3-14C}D(−)-3-hydroxybutyrate

{3-¹⁴C}D(−)-3-hydroxybutyrate or {2-¹⁴C}glucose was injected subcutaneously into 15-day old suckling rats. The animals were killed 3, 6 and 24 hr later by decapitation. Brain proteins, cholesterol, glycolipids, and phospholipids were extracted and prepared for counting. The {3-¹⁴C}D(−)-3-hydroxybutyrate injected animals showed ca. twofold greater labeling (p<.001) of brain cholesterol compared to {2-¹⁴C}glucose; whereas the {2-¹⁴C}glucose injected animals showed ca. fourfold greater labeling (p<.001) of brain proteins than {3-¹⁴C}D(−)-3-hydroxybutyrate at all time points. The difference in labeling of brain glycolipids and phospholipids was less striking, but greater labeling was apparent at each time point in the {2-¹⁴C}glucose injected animals compared to the {3-¹⁴C}D(−)-3-hydroxybutyrate injected animals. These data suggest that D(−)-3-hydroxybutyrate behaves differently than glucose, being a more direct precursor for brain cholesterol biosynthesis and a less effective precursor for brain protein synthesis. Further studies ascertaining the specific activities of the precursors are necessary to quantitatite the respective contributions of D(−)-3-hydroxybutyrate and glucose to lipid and protein synthesis in the rat brain during development.     

Insertion of an elastase-binding loop into interleukin-1{beta}

The protease-binding sequence EAIPMSIPPE from 1-antitrypsinhas been inserted into the cytokine interleykin-1ß,replacing residues 50–53. The resulting mutant proteinwas cleaved specifically at a singly site by elastase and chymotrypsin,but not by trypson. The cleavage by elastase was shown to bebetween Met and Ser of the inserted loop. In contrast, wild-typeinterleukin is not sus-ceptible to cleavage by any of theseenzymes. The mutant protein acts as an inhibitor of elastase,with a K1 of 30 µM. The wild type displays no such inhibitoryactitvity. The overall structure of the mutant, as demonstratedbyu CD, appears to be indistinguishabel from that fo the wildtype. These results indicate that the protease-binding regionfo 1-antitrypson can be recognized and is active even withinthe context of an entirely differentproteinstructure. Giventhat interleukinm-1ß binds to, and is intenalizedby, many types of cells, this hybrid protein also demonstratesthe feasibility of using interleukin-1ß as a deliverysystem for useful therapeutic agents.     

Stepwise transplantation of an active site loop between heat-labile enterotoxins LT-II and LT-I and characterization of the obtained hybrid toxins

Members of the cholera toxin family, including Escherichia coli heat- labile enterotoxins LT-I and LT-II, catalyze the covalent modification of intracellular proteins by transfer of ADP-ribose from NAD to a specific arginine of the target protein. The ADP-ribosylating activity of these toxins is located in the A-subunit, for which LT-I and LT-II share a 63% sequence identity. The flexible loop in LT-I, ranging from residue 47 to 56, closes over the active site cleft. Previous studies have shown that point mutations in this loop have dramatic effects on the activity of LT-I. Yet, in LT-II the sequence of the equivalent loop differs at four positions from LT-I. Therefore five mutants of the active site loop were created by a stepwise replacement of the loop sequence in LT-I with virtually all the corresponding residues in LT- II. Since we discovered that LT-II had no activity versus the artificial substrate diethylamino-benzylidine-aminoguanidine (DEABAG) while LT-I does, our active site mutants most likely probe the NAD binding, not the arginine binding region of the active site. The five hybrid toxins obtained (Q49A, F52N, V53T, Q49V/F52N and Q49V/F52N/V53T) show (i) great differences in holotoxin assembly efficiency; (ii) decreased cytotoxicity in Chinese hamster ovary cells; and (iii) increased in vitro enzymatic activity compared with wild type LT-I. Specifically, the three mutants containing the F52N substitution display a greater Vmax for NAD than wild type LT-I. The enzymatic activity of the V53T mutant is significantly higher than that of wild type LT-I. Apparently this subtle variation at position 53 is beneficial, in contrast to several other substitutions at position 53 which previously had been shown to be deleterious for activity. The most striking result of this study is that the active site loop of LT- I, despite great sensitivity for point mutations, can essentially be replaced by the active site loop of LT-II, yielding an active 'hybrid enzyme' as well as 'hybrid toxin'.      

Comparison of a molecular dynamics model with the X-ray structure of the N370S acid-beta-glucosidase mutant that causes Gaucher disease

Recently, two studies were published that examined the structure of the acid-β-glucosidase N370S mutant, the most common mutant that causes Gaucher disease. One study used the experimental tool of X-ray crystallography, and the other utilized molecular dynamics (MD). The two studies reinforced each other through the similarities in their findings, but each approach also added some unique information. Both studies report that the conformation of active site loop 3 changes, due to an altered hydrogen bonding network; however, the MD study produced additional data concerning the flexibility of loop 1 and the catalytic residues that are not observed in the other study.     

Stabilization of the autoproteolysis of TNF-alpha converting enzyme (TACE) results in a novel crystal form suitable for structure-based drug design studies

The crystallization of TNF-alpha converting enzyme (TACE) has been useful in understanding the structure-activity relationships of new chemical entities. However, the propensity of TACE to undergo autoproteolysis has made enzyme handling difficult and impeded the identification of inhibitor soakable crystal forms. The autoproteolysis of TACE was found to be specific (Y352-V353) and occurred within a flexible loop that is in close proximity to the P-side of the active site. The rate of autoproteolysis was found to be proportional to the concentration of TACE, suggesting a bimolecular reaction mechanism. A limited specificity study of the S(1)' subsite was conducted using surrogate peptides and suggested substitutions that would stabilize the proteolysis of the loop at positions Y352-V353. Two mutant proteases (V353G and V353S) were generated and proved to be highly resistant to autoproteolysis. The kinetics of the more resistant mutant (V353G) and wild-type TACE were compared and demonstrated virtually identical IC(50) values for a panel of competitive inhibitors. However, the k(cat)/K(m) of the mutant for a larger substrate (P6 - P(6)') was approximately 5-fold lower than that for the wild-type enzyme. Comparison of the complexed wild-type and mutant structures indicated a subtle shift in a peripheral P-side loop (comprising the mutation site) that may be involved in substrate binding/turnover and might explain the mild kinetic difference. The characterization of this stabilized form of TACE has yielded an enzyme with similar native kinetic properties and identified a novel crystal form that is suitable for inhibitor soaking and structure determination.     

Incorporation of [1-14C]octadecanol into the lipids ofLeishmania donovani

After incubation of stationary phaseLeishmania donovani with [1-¹⁴C] octadecanol, about 70% of the precursor was taken up within 3 hr. Wax esters and acyl moieties of glycerolipids contained most of the¹⁴C-activity from 3 to 6 hr, because octadecanol was partly oxidized to stearate. Ether moieties were only weakly labeled. After 40 hr, 1-0-aklyl and 1-0-alk-1′-enyl diacylglycerols as well as 1-0-alkyl and 1-0-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamines contained nearly all of the radioactivity. Most of the label in the neutral ether lipids was located in the alkyl ether side chain, whereas, in the phosphatidylethanolamine fraction, most of the label was found in the alkenyl ether side chain. Administration of 1-0-[1-¹⁴C] hexadecyl glycerol resulted in rapid labeling of the vinyl ether side chain of phosphatidylethanolamine plasmalogen (1 hr) increasing further at 2.5 hr. Most of the radioactivity in the alkoxy diacylglycerols was found in the 1-0-alkyl moiety.     

Improving Escherichia coli alkaline phosphatase efficacy by additional mutations inside and outside the catalytic pocket

We describe a strategy that allowed us to confer on a bacterial (E. coli) alkaline phosphatase (AP) the high catalytic activity of the mammalian enzyme while maintaining its high thermostability. First, we identified mutations, at positions other than those occupied by essential catalytic residues, which inactivate the bacterial enzyme without destroying its overall conformation. We transferred concomitantly into the bacterial enzyme four residues of the mammalian enzyme, two being in the catalytic pocket and two being outside. Second, the gene encoding the inactive mutant was submitted to random mutagenesis. Enzyme activity was restored upon the single mutation D330N, at a position that is 12 A away from the center of the catalytic pocket. Third, this mutation was combined with other mutations previously reported to increase AP activity slightly in the presence of magnesium. As a result, at pH 10.0 the phosphatase activity of both mutants D330N/D153H and D330N/D153G was 17-fold higher than that of the wild-type AP. Strikingly, although the two individual mutations D153H and D153G destabilize the enzyme, the double mutant D330N/D153G remained highly stable (T(m)=87 degrees C). Moreover, when combining the phosphatase and transferase activities, the catalytic activity of the mutant D330N/D153G increased 40-fold (k(cat)=3200 s-1) relative to that of the wild-type enzyme (k(cat)=80 s-1). Due to the simultaneous increase in K(m), the resulting k(cat)/K(m) value was only increased by a factor of two. Therefore, a single mutation occurring outside a catalytic pocket can dramatically control not only the activity of an enzyme, but also its thermostability. Preliminary crystallographic data of a covalent D330N/D153G enzyme-phosphate complex show that the phosphate group has significantly moved away from the catalytic pocket, relative to its position in the structure of another mutant previously reported.     

Active-site residues are critical for the folding and stability of methylamine dehydrogenase

Site-directed mutagenesis was used to alter active-site residuesof methylamine dehydrogenase (MADH) from Paracoccus denitrificans.Four residues of the ß subunit of MADH which are inclose proximity to the tryptophan tryptophylquinone (TTQ) prostheticgroup were modified. The crystal structure of MADH reveals thateach of these residues participates in hydrogen bonding interactionswith other active-site residues, TTQ or water. Relatively conservativemutations which removed the potentially reactive oxygens onthe side chains of Thr122, Tyr119, Asp76 and Asp32 each resultedin greatly reduced or undetectable levels of MADH production.The reduction of MADH levels was determined by assays of activityand Western blots of crude extracts with antisera specific forthe MADH ß subunit. No activity or cross-reactiveprotein was detected in extracts of cells expressing D76N, T122Aand T122C MADH mutants. Very low levels of active MADH wereproduced by cells expressing D32N, Y119F, Y119E and Y119K MADHmutants. The Y119F and D32N mutants were purified from cellextracts and found to be significantly less stable than wild-typeMADH. Only the T122S MADH mutant was produced at near wild-typelevels. Possible roles for these amino acid residues in stabilizingunusual structural features of the MADH ß subunit,protein folding and TTQ biosynthesis are discussed.