Tuning of the product spectrum of vanillyl-alcohol oxidase by medium engineering

The flavoenzyme vanillyl-alcohol oxidase (VAO) catalyzes the conversion of 4-alkylphenols through the initial formation of p-quinone methide intermediates. These electrophilic species are stereospecifically attacked by water to yield (R)-1-(4'-hydroxyphenyl)alcohols or rearranged in a competing reaction to 1-(4'-hydroxyphenyl)alkenes. Here, we show that the product spectrum of VAO can be controlled by medium engineering. When the enzymatic conversion of 4-propylphenol was performed in organic solvent, the concentration of the alcohol decreased and the concentration of the cis-alkene, but not the trans-alkene, increased. This change in selectivity occurred in both toluene and acetonitrile and was dependent on the water activity of the reaction medium. A similar shift in alcohol/cis-alkene product ratio was observed when the VAO-mediated conversion of 4-propylphenol was performed in the presence of monovalent anions that bind specifically near the enzyme active site.     

Vanillyl-alcohol oxidase, a tasteful biocatalyst

The covalent flavoenzyme vanillyl-alcohol oxidase (VAO) is a versatile biocatalyst. It converts a wide range of phenolic compounds by catalysing oxidation, deamination, demethylation, dehydrogenation and hydroxylation reactions. The production of natural vanillin, 4-hydroxybenzaldehyde, coniferyl alcohol and enantiomeric pure phenol derivatives is of interest for biotechnological applications. The hydroxylation of 4-alkylphenols is highly stereospecific for the (R)-isomer, whereas dehydrogenation of these substrates is specific for the cis- or trans-isomer. On the basis of crystallographic data, we suggest that the stereospecificity is related to the active site residue Asp170. Another important feature of VAO is the covalent flavin attachment. Studies from site-directed mutants suggest that the covalent flavin–protein interaction improves the catalytic performance as well as the long-term stability of VAO.     

Enantioselective hydroxylation of 4-alkylphenols by vanillyl alcohol oxidase

Vanillyl alcohol oxidase (VAO) from Penicillium simplicissimum catalyzes the enantioselective hydroxylation of 4-ethylphenol, 4-propylphenol, and 2-methoxy-4-propylphenol into 1-(4'-hydroxyphenyl)ethanol, 1-(4'-hydroxyphenyl)propanol, and 1-(4'-hydroxy-3'-methoxyphenyl)propanol, respectively, with an ee of 94% for the R enantiomer. The stereochemical outcome of the reactions was established by comparing the chiral GC retention times of the products to those of chiral alcohols obtained by the action of the lipases from Candida antarctica and Pseudomonas cepacia. Isotope labeling experiments revealed that the oxygen atom incorporated into the alcoholic products is derived from water. During the VAO-mediated conversion of 4-ethylphenol/4-propylphenol, 4-vinylphenol/4-propenylphenol are formed as side products. With 2-methoxy-4-propylphenol as a substrate, this competing side reaction is nearly abolished, resulting in less than 1% of the vinylic product, isoeugenol. The VAO-mediated conversion of 4-alkylphenols also results in small amounts of phenolic ketones indicative for a consecutive oxidation step. Copyright 1998 John Wiley & Sons, Inc.     

Regio- and Stereospecific Conversion of 4-Alkylphenols by the Covalent Flavoprotein Vanillyl-Alcohol Oxidase

The regio- and stereospecific conversion of prochiral 4-alkylphenols by the covalent flavoprotein vanillyl-alcohol oxidase was investigated. The enzyme was active, with 4-alkylphenols bearing aliphatic side chains of up to seven carbon atoms. Optimal catalytic efficiency occurred with 4-ethylphenol and 4-n-propylphenols. These short-chain 4-alkylphenols are stereoselectively hydroxylated to the corresponding (R)-1-(4′-hydroxyphenyl)alcohols (F. P. Drijfhout, M. W. Fraaije, H. Jongejan, W. J. H. van Berkel, and M. C. R. Franssen, Biotechnol. Bioeng. 59:171–177, 1998). (S)-1-(4′-Hydroxyphenyl)ethanol was found to be a far better substrate than (R)-1-(4′-hydroxyphenyl)ethanol, explaining why during the enzymatic conversion of 4-ethylphenol nearly no 4-hydroxyacetophenone is formed. Medium-chain 4-alkylphenols were exclusively converted by vanillyl-alcohol oxidase to the corresponding 1-(4′-hydroxyphenyl)alkenes. The relative cis-trans stereochemistry of these reactions was strongly dependent on the nature of the alkyl side chain. The enzymatic conversion of 4-sec-butylphenol resulted in two (4′-hydroxyphenyl)-sec-butene isomers with identical masses but different fragmentation patterns. We conclude that the water accessibility of the enzyme active site and the orientation of the hydrophobic alkyl side chain of the substrate are of major importance in determining the regiospecific and stereochemical outcome of vanillyl-alcohol oxidase-mediated conversions of 4-alkylphenols.     

Alkylphenol biotransformations catalyzed by 4-ethylphenol methylenehydroxylase

4-ethylphenol methylenehydroxylase from Pseudomonas putida JD1 acts by dehydrogenation of its substrate to give a quinone methide, which is then hydrated to an alcohol. It was shown to be active with a range of 4-alkylphenols as substrates. 4-n-propylphenol, 4-n-butylphenol, chavicol, and 4-hydroxydiphenylmethane were hydroxylated on the methylene group next to the benzene ring and produced the corresponding chiral alcohol as the major product. The alcohols 1-(4'-hydroxyphenyl)propanol and 1-(4'-hydroxyphenyl)-2-propen-1-ol, produced by the biotransformation of 4-n-propylphenol and chavicol, respectively, were shown to be R(+) enantiomers. 5-Indanol, 6-hydroxytetralin, 4-isopropylphenol, and cyclohexylphenol, with cyclic or branched alkyl groups, gave the corresponding vinyl compounds as their major products.     

Insights into the catalytic roles of the polypeptide regions in the active site of thermolysin and generation of the thermolysin variants with high activity and stability

The active site of thermolysin is composed of one zinc ion and five polypeptide regions [N-terminal sheet (Asn112-Trp115), alpha-helix 1 (Val139-Thr149), C-terminal loop 1 (Asp150-Gly162), alpha-helix 2 (Ala163-Val176) and C-terminal loop 2 (Gln225-Ser234)]. To explore their catalytic roles, we introduced single amino-acid substitutions into these regions by site-directed mutagenesis and examined their effects on the activity and stability. Seventy variants, in which one of the twelve residues (Ala113, Phe114, Trp115, Asp150, Tyr157, Gly162, Ile168, Ser169, Asp170, Asn227, Val230 and Ser234) was replaced, were produced in Escherichia coli. The hydrolytic activities of thermolysin for N-[3-(2-furyl)acryloyl]-Gly-l-Leu amide (FAGLA) and casein revealed that the N-terminal sheet and alpha-helix 2 were critical in catalysis and the C-terminal loops 1 and 2 were in substrate recognition. Twelve variants were active for both substrates. In the hydrolysis of FAGLA and N-carbobenzoxy-L-Asp-L-Phe methyl ester, the k(cat)/K(m) values of the D150E (in which Asp150 is replaced with Glu) and I168A variants were 2-3 times higher than those of the wild-type (WT) enzyme. Thermal inactivation of thermolysin at 80 degrees C was greatly suppressed with the D150H, D150W, I168A, I168H, N227A, N227H and S234A. The evidence might provide the insights into the activation and stabilization of thermolysin.     

Characterization of rat and mouse NAD+-dependent 3alpha/17beta/20alpha-hydroxysteroid dehydrogenases and identification of substrate specificity determinants by site-directed mutagenesis

In this study, we characterized rat and mouse aldo-keto reductases (AKR1C16 and AKR1C13, respectively) with 92% sequence identity. The recombinant enzymes oxidized non-steroidal alcohols using NAD⁺ as the preferred coenzyme, and showed low 3α/17β/20α-hydroxysteroid dehydrogenase (HSD) activities. The substrate specificity differs from that of rat NAD⁺-dependent 3α-HSD (AKR1C17) that shares 95% sequence identity with AKR1C16. To elucidate the residues determining the substrate specificity of the enzymes, we performed site-directed mutagenesis of Tyr24, Asp128 and Phe129 of AKR1C16 with the corresponding residues (Ser, Tyr and Leu, respectively) of AKR1C17. The double mutation (Asp128/Tyr-Phe129/Leu) had few effects on the substrate specificity, while the Tyr24/Ser mutant showed only 3α-HSD activity, and the triple mutation of the three residues produced an enzyme that had almost the same properties as AKR1C17. The importance of the residue 24 for substrate recognition was verified by the mutagenesis of Ser24/Tyr of AKR1C17 which resulted in a decrease in 3α-HSD activity and appearance of 17β- and 20α-HSD activities. AKR1C16 is also 92% identical with rat NAD⁺-dependent 17β-HSD (AKR1C24), which possesses Tyr24. The replacement of Asp128, Phe129 and Ser137 of AKR1C16 with the corresponding residues (Glu, Ser and Phe, respectively) of AKR1C24 increased the catalytic efficiency for 17β- and 20α-hydroxysteroids.     

Conformational rearrangement of beta-lactoglobulin upon interaction with an anionic membrane

The recent availability of the SHV-1 beta-lactamase crystal structure provides a framework for the understanding of the functional role of amino acid residues in this enzyme. To that end, we have constructed by site-directed mutagenesis 18 variants of the SHV beta-lactamase: an extended spectrum group: Gly238Ser, Gly238Ser-Glu240Lys, Asp104Lys-Gly238Ser, Asp104Lys-Thr235Ser-Gly238Ser, Asp179Asn, Arg164His, and Arg164Ser; an inhibitor resistant group: Arg244Ser, Met69Ile, Met69Leu, and Ser130Gly; mutants that are synergistic with those that confer resistance to oxyimino-cephalosporins: Asp104Glu, Asp104Lys, Glu240Lys, and Glu240Gln; and structurally conserved mutants: Thr235Ser, Thr235Ala and Glu166Ala. Among the extended spectrum group the combination of high-level ampicillin and cephalosporin resistance was demonstrated in the Escherichia coli DH10B strains possessing the Gly238Ser mutation: Gly238Ser, Gly238Ser-Glu240Lys, Asp104Lys-Gly238Ser, and Asp104Lys-Thr235Ser-Gly238Ser. Of the inhibitor resistant group, the Ser130Gly mutant was the most resistant to ampicillin/clavulanate. Using a polyclonal anti-SHV antibody, we assayed steady state protein expression levels of the SHV beta-lactamase variants. Mutants with the Gly238Ser substitution were among the most highly expressed. The Gly238Ser substitution resulted in an improved relative k(cat)/K(m) value for cephaloridine and oxyimino-cephalosporins compared to SHV-1 and Met69Ile. In our comparative survey, the Gly238Ser and extended spectrum beta-lactamase variants containing this substitution exhibited the greatest substrate versatility against penicillins and cephalosporins and greatest protein expression. This defines a unique role of Gly238Ser in broad-spectrum beta-lactam resistance in this family of class A beta-lactamases.     

Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis.

Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family. We have analyzed the roles of the conserved residues in this enzyme (His103, Glu128, Gly163, Asp164, Ser165, Gly167, Asp262, Asp266 and His292) by site-directed mutagenesis. Among them, Gly163, Asp164, Ser165, and Gly167 are the components of a G-D/E-S-A-G motif. We showed that Ser165, Asp262, and His292 are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp164, is critical for the enzymatic activity. The mutation of Asp164 to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding. This residue is probably structurally important to make the conformation of the active-site functional.     

Identification of residues in the carboxy-terminal domain of Clostridium perfringens alpha-toxin (phospholipase C) which are required for its biological activities

A panel of random mutants within the DNA encoding the carboxy-terminal domain of Clostridium perfringens alpha-toxin was constructed. Three mutants were identified which encoded alpha-toxin variants (Lys330Glu, Asp305Gly, and Asp293Ser) with reduced hemolytic activity. These variants also had diminished phospholipase C activity toward aggregated egg yolk phospholipid and reduced cytotoxic and myotoxic activities. Asp305Gly showed a significantly increased enzymatic activity toward the monodisperse substrate rhoNPPC, whereas Asp293Ser displayed a reduced activity toward this phospholipid analogue. In addition, Asp293Ser showed an increased dependence on calcium for enzymatic activity toward aggregated phospholipid and appeared calcium-depleted in PAGE band-shift assays. In contrast, neither Lys330Glu nor Asp305Gly showed altered dependence on calcium for enzymatic activity toward aggregated phospholipid. Asp305 is located in the interface between the amino- and carboxy-terminal domains, whereas Asp293 and Lys330 are surface exposed residues which may play a role in the recognition of membrane phospholipids.     

Catalytic mechanism of phosphorylase kinase probed by mutational studies.

The contributions to catalysis of the conserved catalytic aspartate (Asp149) in the phosphorylase kinase catalytic subunit (PhK; residues 1-298) have been studied by kinetic and crystallographic methods. Kinetic studies in solvents of different viscosity show that PhK, like cyclic AMP dependent protein kinase, exhibits a mechanism in which the chemical step of phosphoryl transfer is fast and the rate-limiting step is release of the products, ADP and phosphoprotein, and possibly viscosity-dependent conformational changes. Site-directed mutagenesis of Asp149 to Ala and Asn resulted in enzymes with a small increase in K(m) for glycogen phosphorylase b (GPb) and ATP substrates and dramatic decreases in k(cat) (1.3 x 10(4) for Asp149Ala and 4.7 x 10(3) for Asp149Asn mutants, respectively). Viscosometric kinetic measurements with the Asp149Asn mutant showed a reduction in the rate-limiting step for release of products by 4.5 x 10(3) and a significant decrease (possibly as great as 2.2 x 10(3)) in the rate constant characterizing the chemical step. The date combined with the crystallographic evidence for the ternary PhK-AMPPNP-peptide complex [Lowe et al. (1997) EMBO J. 6, 6646-6658] provide powerful support for the role of the carboxyl of Asp149 in binding and orientation of the substrate and in catalysis of phosphoryl transfer. The constitutively active subunit PhK has a glutamate (Glu182) residue in the activation segment, in place of a phosphorylatable serine, threonine, or tyrosine residue in other protein kinases that are activated by phosphorylation. Site-directed mutagenesis of Glu182 and other residues involved in a hydrogen bond network resulted in mutant proteins (Glu182Ser, Arg148Ala, and Tyr206Phe) with decreased catalytic efficiency (approximate average decrease in k(cat)/K(m) by 20-fold). The crystal structure of the mutant Glu182Ser at 2.6 A resolution showed a phosphate dianion about 2.6 A from the position previously occupied by the carboxylate of Glu182. There was no change in tertiary structure from the native protein, but the activation segment in the region C-terminal to residue 182 showed increased disorder, indicating that correct localization of the activation segment is necessary in order to recognize and present the protein substrate for catalysis.     

Electrostatic repulsion, compensatory mutations, and long-range non-additive effects at the dimerization interface of the HIV capsid protein

In previous studies, thermodynamic dissection of the dimerization interface in CA-C, the C-terminal domain of the capsid protein of human immunodeficiency virus type 1, revealed that individual mutation to alanine of Ser178, Glu180, Glu187 or Gln192 led to significant increases in dimerization affinity. Four related aspects derived from this observation have been now addressed, and the results can be summarized as follows: (i) thermodynamic analyses indicate the presence of an intersubunit electrostatic repulsion between both Glu180 residues. (ii) The mutation Glu180 to Ala was detected in nearly all type 2 human immunodeficiency virus variants, and in several simian immunodeficiency viruses analyzed. However, this mutation was strictly co-variant with mutations Ser178Asp in a neighboring residue, and Glu187Gln. Thermodynamic analysis of multiple mutants showed that Ser178Asp compensated, alone or together with Glu187Gln, the increase in affinity caused by the mutation Glu180Ala, and restored a lower dimerization affinity. (iii) The increase in the affinity constant caused by the multiple mutation to Ala of Ser178, Glu180, Glu187 and Gln192 was more than one order of magnitude lower than predicted if additivity were present, despite the fact that the 178/180 pair and the two other residues were located more than 10A apart. (iv) Mutations in CA-C that caused non-additive increases in dimerization affinity also caused a non-additive increase in the capacity of the isolated CA-C domain to inhibit the assembly of capsid-like HIV-1 particles in kinetic assays. In summary, the study of a protein-protein interface involved in the building of a viral capsid has revealed unusual features, including intersubunit electrostatic repulsions, co-variant, compensatory mutations that may evolutionarily preserve a low association constant, and long-range, large magnitude non-additive effects on association.     

Metabolism of some amino acids in relation to the photorespiratory nitrogen cycle of pea leaves

¹⁵N-labelled (amino group) asparagine (Asn), glutamate (Glu), alanine (Ala), aspartate (Asp) and serine (Ser) were used to study the metabolic role and the participation of each compound in the photorespiratory N cycle ofPisum sativum L. leaves. Asparagine was utilised as a nitrogen source by either deamidation or transamination, Glu was converted to Gln through NH₃ assimilation and was a major amino donor for transamination, and Ala was utilised by transamination to a range of amino acids. Transamination also provided a pathway for Asp utilisation, although Asp was also used as a substrate for Asn synthesis. In the photorespiratory synthesis of glycine (Gly), Ser, Ala, Glu and Asn acted as sources of amino-N, contributing, in the order given, 38, 28, 23, and 7% of the N for glycine synthesis; Asp provided less than 4% of the amino-N in glycine. Calculations based on the incorporation of¹⁵N into Gly indicated that about 60% (Ser), 20% (Ala), 12% (Glu) and 11% (Asn) of the N metabolised from each amino acid was utilised in the photorespiratory nitrogen cycle.Abbreviations Ala alamine - Asn asparagine - Asp aspartate - Glu glutamate - MOA methoxylamine - Ser serine     

Engineering subtilisin E for enhanced stability and activity in polar organic solvents

We examined the effect of a novel disulfide bond engineered in subtilisin E from Bacillus subtilis based on the structure of a thermophilic subtilisin-type serine protease aqualysin I. Four sites (Ser163/Ser194, Lys170/Ser194, Lys170/Glu195, and Pro172/Glu195) in subtilisin E were chosen as candidates for Cys substitutions by site-directed mutagenesis. The Cys170/Cys195 mutant subtilisin formed a disulfide bond in B. subtilis, and showed a 5-10-fold increase in specific activity for an authentic peptide substrate for subtilisin, N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide, compared with the single-Cys mutants. However, the disulfide mutant had a 50% decrease in catalytic efficiency due to a smaller k(cat) and was thermolabile relative to the wild-type enzyme, whereas it was greatly stabilized relative to its reduced form. These results suggest that an electrostatic interaction between Lys170 and Glu195 is important for catalysis and stability in subtilisin E. Interestingly, the disulfide mutant was found to be more stable in polar organic solvents, such as dimethylformamide and ethanol, than the wild-type enzyme, even under reducing conditions; this is probably due to the substitution of uncharged Cys by charged surface residues (Lys170 and Glu195). Further, the amino-terminal engineered disulfide bond (Gly61Cys/Ser98Cys) and the mutation Ile31Leu were introduced to enhance the stability and catalytic activity. A prominent 3-4-fold increase in the catalytic efficiency occurred in the quintet mutant enzyme over the range of dimethylformamide concentration (up to 40%).     

Heparan/chondroitin sulfate biosynthesis. Structure and mechanism of human glucuronyltransferase I

Human beta1,3-glucuronyltransferase I (GlcAT-I) is a central enzyme in the initial steps of proteoglycan synthesis. GlcAT-I transfers a glucuronic acid moiety from the uridine diphosphate-glucuronic acid (UDP-GlcUA) to the common linkage region trisaccharide Gal beta 1-3Gal beta 1-4Xyl covalently bound to a Ser residue at the glycosaminylglycan attachment site of proteoglycans. We have now determined the crystal structure of GlcAT-1 at 2.3 A in the presence of the donor substrate product UDP, the catalytic Mn(2+) ion, and the acceptor substrate analog Gal beta 1-3Gal beta 1-4Xyl. The enzyme is a alpha/beta protein with two subdomains that constitute the donor and acceptor substrate binding site. The active site residues lie in a cleft extending across both subdomains in which the trisaccharide molecule is oriented perpendicular to the UDP. Residues Glu(227), Asp(252), and Glu(281) dictate the binding orientation of the terminal Gal-2 moiety. Residue Glu(281) is in position to function as a catalytic base by deprotonating the incoming 3-hydroxyl group of the acceptor. The conserved DXD motif (Asp(194), Asp(195), Asp(196)) has direct interaction with the ribose of the UDP molecule as well as with the Mn(2+) ion. The key residues involved in substrate binding and catalysis are conserved in the glucuronyltransferase family as well as other glycosyltransferases.     

The N-terminal nucleophile serine of cephalosporin acylase executes the second autoproteolytic cleavage and acylpeptide hydrolysis

Cephalosporin acylase (CA) precursor is translated as a single polypeptide chain and folds into a self-activating pre-protein. Activation requires two peptide bond cleavages that excise an internal spacer to form the mature αβ heterodimer. Using Q-TOF LC-MS, we located the second cleavage site between Glu(159) and Gly(160), and detected the corresponding 10-aa spacer (160)GDPPDLADQG(169) of CA mutants. The site of the second cleavage depended on Glu(159): moving Glu into the spacer or removing 5-10 residues from the spacer sequence resulted in shorter spacers with the cleavage at the carboxylic side of Glu. The mutant E159D was cleaved more slowly than the wild-type, as were mutants G160A and G160L. This allowed kinetic measurements showing that the second cleavage reaction was a first-order, intra-molecular process. Glutaryl-7-aminocephalosporanic acid is the classic substrate of CA, in which the N-terminal Ser(170) of the β-subunit, is the nucleophile. Glu and Asp resemble glutaryl, suggesting that CA might also remove N-terminal Glu or Asp from peptides. This was indeed the case, suggesting that the N-terminal nucleophile also performed the second proteolytic cleavage. We also found that CA is an acylpeptide hydrolase rather than a previously expected acylamino acid acylase. It only exhibited exopeptidase activity for the hydrolysis of an externally added peptide, supporting the intra-molecular interaction. We propose that the final CA activation is an intra-molecular process performed by an N-terminal nucleophile, during which large conformational changes in the α-subunit C-terminal region are required to bridge the gap between Glu(159) and Ser(170).     

Effect on catalysis by replacement of catalytic residue from hen egg white lysozyme to Venerupis philippinarum lysozyme*

Asn46Asp/Asp52Ser or Asn46Glu/Asp52Ser hen egg white lysozyme (HEL) mutant was designed by introducing the substituted catalytic residue Asp46 or Glu46, respectively, based on Venerupis philippinarum (Vp) lysozyme structure as a representative of invertebrate‐type (i‐type) lyzozyme. These mutations restored the bell‐shaped pH‐dependency of the enzyme activity from the sigmoidal pH‐dependency observed for the Asp52Ser mutant. Furthermore both lysozyme mutants possessed retaining mechanisms like Vp lysozyme and HEL. The Asn46Glu/Asp52Ser mutant, which has a shorter distance between two catalytic residues, formed a glycosyl adduct in the reaction with the N‐acetylglucosamine oligomer. Furthermore, we found the accelerated turnover through its glycosyl adduct formation and decomposition. The turnover rate estimated from the glycosyl formation and decomposition rates was only 20% of the observed hydrolysis rate of the substrate. Based on these results, we discussed the catalytic mechanism of lysozymes.     

Mitochondrial phosphate transport protein. Reversions of inhibitory conservative mutations identify four helices and a nonhelix protein segment with transmembrane interactions and Asp39, Glu137, and Ser158 as nonessential for transport

The mitochondrial phosphate transport protein (PTP) has six (A--F) transmembrane (TM) helices per subunit of functional homodimer with all mutations referring to the subunit of the homodimer. In earlier studies, conservative replacements of several residues located either at the matrix end (Asp39/helix A, Glu137/helix C, Asp236/helix E) or at the membrane center (His32/helix A, Glu136/helix C) of TM helices yielded inactive single mutation PTPs. Some of these residues were suggested to act as phosphate ligands or as part of the proton cotransport path. We now show that the mutation Ser158Thr, not part of a TM helix but located near the center of the matrix loop (Ile141--Ser171) between TM helices C and D, inactivates PTP and is thus also functionally relevant. On the other side of the membrane, the single mutation Glu192Asp at the intermembrane space end of TM helix D yields a PTP with 33% wild-type activity. We constructed double mutants by adding this mutation to the six transport-inactivating mutations. Transport was detected only in those with Asp39Asn, Glu137Gln, or Ser158Thr. We conclude that TM helix D can interact with TM helices A and C and matrix loop Ile141--Ser171 and that Asp39, Glu137, and Ser158 are not essential for phosphate transport. Since our results are consistent with residues present in all 12 functionally identified members of the mitochondrial transport protein (MTP) family, they lead to a general rule that specifies MTP residue types at 7 separate locations. The conformations of all the double mutation PTPs (except that with the matrix loop Ser158Thr) are significantly different from those of the single mutation PTPs, as indicated by their very low liposome incorporation efficiency and their requirement for less detergent (Triton X-100) to stay in solution. These dramatic conformational differences also suggest an interaction between TM helices D and E. The results are discussed in terms of TM helix movements and changes in the PTP monomer/dimer ratio.     

Two CES1 gene mutations lead to dysfunctional carboxylesterase 1 activity in man: clinical significance and molecular basis

The human carboxylesterase 1 (CES1) gene encodes for the enzyme carboxylesterase 1, a serine esterase governing both metabolic deactivation and activation of numerous therapeutic agents. During the course of a study of the pharmacokinetics of the methyl ester racemic psychostimulant methylphenidate, profoundly elevated methylphenidate plasma concentrations, unprecedented distortions in isomer disposition, and increases in hemodynamic measures were observed in a subject of European descent. These observations led to a focused study of the subject's CES1 gene. DNA sequencing detected two coding region single-nucleotide mutations located in exons 4 and 6. The mutation in exon 4 is located in codon 143 and leads to a nonconservative substitution, p.Gly143Glu. A deletion in exon 6 at codon 260 results in a frameshift mutation, p.Asp260fs, altering residues 260-299 before truncating at a premature stop codon. The minor allele frequency of p.Gly143Glu was determined to be 3.7%, 4.3%, 2.0%, and 0% in white, black, Hispanic, and Asian populations, respectively. Of 925 individual DNA samples examined, none carried the p.Asp260fs, indicating it is an extremely rare mutation. In vitro functional studies demonstrated the catalytic functions of both p.Gly143Glu and p.Asp260fs are substantially impaired, resulting in a complete loss of hydrolytic activity toward methylphenidate. When a more sensitive esterase substrate, p-nitrophenyl acetate was utilized, only 21.4% and 0.6% catalytic efficiency (V(max)/K(m)) were determined in p.Gly143Glu and p.Asp260fs, respectively, compared to the wild-type enzyme. These findings indicate that specific CES1 gene variants can lead to clinically significant alterations in pharmacokinetics and drug response of carboxylesterase 1 substrates.     

Contributions of long-range electrostatic interactions to 4-chlorobenzoyl-CoA dehalogenase catalysis: a combined theoretical and experimental study

It is well established that electrostatic interactions play a vital role in enzyme catalysis. In this work, we report theory-guided mutation experiments that identified strong electrostatic contributions of a remote residue, namely, Glu232 located on the adjacent subunit, to 4-chlorobenzoyl-CoA dehalogenase catalysis. The Glu232Asp mutant was found to bind the substrate analogue 4-methylbenzoyl-CoA more tightly than does the wild-type dehalogenase. In contrast, the kcat for 4-chlorobenzoyl-CoA conversion to product was reduced 10000-fold in the mutant. UV difference spectra measured for the respective enzyme-ligand complexes revealed an approximately 3-fold shift in the equilibrium of the two active site conformers away from that inducing strong pi-electron polarization in the ligand benzoyl ring. Increased substrate binding, decreased ring polarization, and decreased catalytic efficiency indicated that the repositioning of the point charge in the Glu232Asp mutant might affect the orientation of the Asp145 carboxylate with respect to the substrate aromatic ring. The time course for formation and reaction of the arylated enzyme intermediate during a single turnover was measured for wild-type and Glu232Asp mutant dehalogenases. The accumulation of arylated enzyme in the wild-type dehalogenase was not observed in the mutant. This indicates that the reduced turnover rate in the mutant is the result of a slow arylation of Asp145, owing to decreased efficiency in substrate nucleophilic attack by Asp145. To rationalize the experimental observations, a theoretical model is proposed, which computes the potential of mean force for the nucleophilic aromatic substitution step using a hybrid quantum mechanical/molecular mechanical method. To this end, the removal or reorientation of the side chain charge of residue 232, modeled respectively by the Glu232Gln and Glu232Asp mutants, is shown to increase the rate-limiting energy barrier. The calculated 23.1 kcal/mol free energy barrier for formation of the Meisenheimer intermediate in the Glu232Asp mutant represents an increase of 6 kcal/mol relative to that of the wild-type enzyme, consistent with the 5.6 kcal/mol increase calculated from the difference in experimentally determined rate constants. On the basis of the combination of the experimental and theoretical evidence, we hypothesize that the Glu232(B) residue contributes to catalysis by providing an electrostatic force that acts on the Asp145 nucleophile.