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BBA - Proteins and Proteomics (v.1774, #12)

Editorial Board (pp. ii).

3D structure of Syk kinase determined by single-particle electron microscopy by Ernesto Arias-Palomo; María A. Recuero-Checa; Xosé R. Bustelo; Oscar Llorca (pp. 1493-1499).

The cytoplasmic Syk kinase plays key roles in immune responses and comprises two N-terminal regulatory Src homology 2 (SH2) domains followed by a catalytic region. Atomic structures of these domains have only been solved in isolation. To gain insights into the three-dimensional structure of full-length Syk, we have used single-particle electron microscopy. Syk acquires a closed conformation resembling the inhibited structure of Zap-70, another member of the Syk family. Such configuration suggests an inhibition of the N-terminal domains on its catalytic activity. The phosphotyrosine binding pockets of both SH2 domains are not occluded and they could interact with other phosphoproteins.

Keywords: Single-particle electron microscopy; EM; Syk; Zap-70; Kinases

Salt effects on β-glucosidase: pH-profile narrowing by Erin M. Bowers; Lindsey O. Ragland; Larry D. Byers (pp. 1500-1507).

Salts inhibit the activity of sweet almond β-glucosidase. For cations (Cl⁻ salts) the effectiveness follows the series: Cu⁺², Fe⁺²>Zn⁺²>Li⁺>Ca⁺²>Mg⁺²>Cs⁺>NH₄⁺>Rb⁺>K⁺>Na⁺ and for anions (Na⁺ salts) the series is: I⁻>ClO₄⁻>⁻SCN>Br⁻≈NO₃⁻>Cl⁻≈⁻OAc>F⁻≈SO₄−². The activity of the enzyme, like that of most glycohydrolases, depends on a deprotonated carboxylate (nucleophile) and a protonated carboxylic acid for optimal activity. The resulting pH-profile of k_cat/ K_m for the β-glucosidase-catalyzed hydrolysis of p-nitrophenyl glucoside is characterized by a width at half height that is strongly sensitive to the nature and concentration of the salt. Most of the inhibition is due to a shift in the enzymic p K_as and not to an effect on the pH-independent second-order rate constant, ( k_cat/ K_m)^lim. For example, as the NaCl concentration is increased from 0.01 M to 1.0 M the apparent p K_a1 increases (from 3.7 to 4.9) and the apparent p K_a2 decreases (from 7.2 to 5.9). With p-nitrophenyl glucoside, the value of the pH-independent ( k_cat/ K_m)^lim (=9×10⁴ M−¹ s−¹) is reduced by less than 4% as the NaCl concentration is increased. There is a similar shift in the p K_as when the LiCl concentration is increased to 1.0 M. The results of these salt-induced p K_a shifts rule out a significant contribution of reverse protonation to the catalytic efficiency of the enzyme. At low salt concentration, the fraction of the catalytically active monoprotonated enzyme in the reverse protonated form (i.e., proton on the group with a p K_a of 3.7 and dissociated from the group with a p K_a of 7.2) is very small (≈0.03%). At higher salt concentrations, where the two p K_as become closer, the fraction of the monoprotonated enzyme in the reverse protonated form increases over 300-fold. However, there is no increase in the intrinsic reactivity, ( k_cat/ K_m)^lim, of the monoprotonated species. For other enzymes which may show such salt-induced p K_a shifts, this provides a convenient test for the role of reverse protonation.

Keywords: Abbreviations; HEPES; N; -(2-hydroxyethyl)-piperazine-; N'; -(2-ethanesulfonate); MES; 2-(; N; -morpholino)ethanesulfonate; PIPES; piperazine-; N; ,; N'; -bis(2-ethanesulfonate); pNPG; p; -nitrophenyl β-; d; -glucopyranosideβ-glucosidase; LiCl; Methyl β-glucoside; pH; Salts

Characterization of the metallocenter of rabbit skeletal muscle AMP deaminase. A new model for substrate interactions at a dinuclear cocatalytic Zn site by Daniela Martini; Maria Ranieri-Raggi; Antonietta R.M. Sabbatini; Arthur J.G. Moir; Enza Polizzi; Stefano Mangani; Antonio Raggi (pp. 1508-1518).

We have previously provided evidence for a dinuclear zinc site in rabbit skeletal muscle AMPD compatible with a (μ-aqua)(μ-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site. XAS of the zinc binding site of the enzyme in the presence of PRN favors a model where PRN is added to the coordination sphere of one of the two zinc ions increasing its coordination number to five. The uncompetitive nature of the inhibition of AMPD by fluoride reveals that the anion probably displaces the nucleophile water molecule terminally coordinated to the catalytic Zn₁ ion at the enzyme C-terminus, following the binding of AMP at the Zn₂ ion located at N-terminus of the enzyme. Thus, the two Zn ions in the AMPD metallocenter operate together as a single catalytic unit, but have independent function, one of them (Zn₁) acting to polarize the nucleophile water molecule, whilst the other (Zn₂) acts transiently as a receptor for an activating substrate molecule. The addition of fluoride to AMPD also abolishes the cooperative behaviour induced in the enzyme by the inhibitory effect of ATP at acidic pH that probably resides in the competition with the substrate for an adenine nucleotide specific regulatory site located in the Zn₂ ion binding region and which is responsible for the positive homotropic cooperativity behaviour of AMPD.

Keywords: Abbreviations; AMPD; AMP deaminase; ADA; adenosine deaminase; HPRG; histidine-proline-rich-glycoprotein; PRN; purine ribonucleoside; EXAFS; Extended X-ray Absorption Fine Structure; FT; Fourier Transform; TNBS; trinitrobenzene sulfonic acid; XANES; X-ray Absorption Near Edge Structure; XAS; X-ray Absorption spectroscopy; BVS; bond valence sum; FAC1; Arabidopsis embryonic factor 1AMP deaminase; Purine ribonucleoside; Zinc binding site; X-ray absorption spectroscopy

Changing the substrate specificity of creatine kinase from creatine to glycocyamine: Evidence for a highly evolved active site by Michael J. Jourden; Callisia N. Clarke; Allyson K. Palmer; Emily J. Barth; Rebecca C. Prada; Robyn N. Hale; Dean Fraga; Mark J. Snider; Paul L. Edmiston (pp. 1519-1527).

Eight variants of creatine kinase were created to switch the substrate specificity from creatine to glycocyamine using a rational design approach. Changes to creatine kinase involved altering several residues on the flexible loops that fold over the bound substrates including a chimeric replacement of the guanidino specificity loop from glycocyamine kinase into creatine kinase. A maximal 2000-fold change in substrate specificity was obtained as measured by a ratio of enzymatic efficiency ( k_cat/ K_M· K_d) for creatine vs. glycocyamine. In all cases, a change in specificity was accompanied by a large drop in enzymatic efficiency. This data, combined with evidence from other studies, indicate that substrate specificity in the phosphagen kinase family is obtained by precise alignment of substrates in the active site to maximize k_cat/ K_M· K_d as opposed to selective molecular recognition of one guanidino substrate over another. A model for the evolution of the dimeric forms of phosphagen kinases is proposed in which these enzymes radiated from a common ancestor that may have possessed a level of catalytic promiscuity. As mutational events occurred leading to greater degrees of substrate specificity, the dimeric phosphagen kinases became evolutionary separated such that the substrate specificity could not be interchanged by a small number of mutations.

Keywords: Creatine kinase; Substrate specificity; Phosphagen kinase evolution

Stabilized recombinant suppressors of RNA silencing: Functional effects of linking monomers of Carnation Italian Ringspot virus p19 by Jenny Cheng; Selena M. Sagan; Naila Assem; Roger Koukiekolo; Natalie K. Goto; John Paul Pezacki (pp. 1528-1535).

Eukaryotes have evolved complex cellular responses to double-stranded RNA including the RNA silencing pathway. Tombusviruses have adapted a mechanism to evade RNA silencing that involves a 19 kDa dimeric protein (p19) that is a suppressor of RNA silencing. In order to develop stabilized p19 proteins, linked versions of p19 from the Carnation Italian Ringspot virus (CIRV) were constructed that joined the C-terminus of one subunit to the N-terminus of the second subunit. Like the native CIRV p19, these linked p19 proteins were able to bind to double-stranded siRNAs with nanomolar affinity and discriminate siRNA according to length. In addition, the interdomain linker improved both the stability and binding properties of the p19 dimer. The observed binding properties support the idea that the semi-rigid cross-link favors the folded, binding-competent state of p19. The cross-linked recombinant CIRV-p19s represent novel stabilized suppressors of RNA silencing and may be useful in future biophysical, immunological and cell biology studies.

Keywords: p19; siRNA; RNA silencing; Linked dimer

Transport proteins PotD and Crr of Escherichia coli, novel fusion partners for heterologous protein expression by Kyung-Yeon Han; Hyuk-Seong Seo; Jong-Am Song; Keum-Young Ahn; Jin-Seung Park; Jeewon Lee (pp. 1536-1543).

The Escherichia coli proteome response to the stressor GdnHCl was analyzed through 2-dimensional gel electrophoresis (2-DE). We identified PotD (spermidine/putrescine-binding periplasmic protein) and Crr [glucose-specific phosphotransferase (PTS) enzyme IIA component] as a stress-responsive protein. Even under a stress situation where the total number of soluble proteins decreased by about 10%, 3.5- and 2.2-fold increase was observed in the synthesis of PotD and Crr, respectively. As fusion partners, PotD and Crr dramatically increased the solubility of many aggregation-prone heterologous proteins [e.g. human minipro-insulin (mp-INS), human epidermal growth factor (EGF), human prepro-ghrelin (ppGRN), human interleukin-2(hIL-2), human activation induced cytidine deaminase (AID), human glutamate decarboxylase (GAD_448–585), Pseudomonas putida cutinase (CUT), human ferritin light chain (hFTN-L), human granulocyte colony-stimulating factor (G-CSF), and cold autoinflammatory syndrome1 protein (NALP3) Nacht domain (NACHT)] in the E. coli cytoplasm. Presumably PotD and Crr were very effective in shielding interactive surfaces of heterologous proteins associated with non-specific protein–protein interactions leading to the formation of inclusion bodies most likely due to intrinsic high folding efficiency, chaperone-like activity, or a combination of both factors. Both the stress-induced proteins were well suited for the production of a biologically active fusion mutant of P. putida cutinase that can be expected to be of biotechnological and commercial interest.

Keywords: Escherichia coli; BL21 proteome; Stress response; PotD; Crr; Stress-responsive protein; Solubility enhancer

Modulation of cutinase stability and structure by phospholipid detergents by Pankaj Sehgal; Søren Bang Nielsen; Shona Pedersen; Reinhard Wimmer; Daniel E. Otzen (pp. 1544-1554).

Fusarium solani pisi cutinase hydrolyses triglycerides of different lengths. Here we show that micelle-forming short-chain (C6–C9) phospholipids significantly reduce cutinase stability (both below and above the critical micelle concentration cmc) and rates of folding (only above cmc), trapping cutinase in an inactive state which only regains activity over hours to days, rather than the few seconds required for refolding in the absence of detergent. Destabilization decreases with increasing chain length, and increases with cmc, indicating that monomers and micelles cooperate in destabilizing cutinase. Detergents have little effect on enzymatic activity and confer no changes in secondary structure. Some changes in chemical shift occur around the enzyme active site, although distant regions are also affected. To our knowledge, this is the first example of marked destabilization of a water-soluble protein by zwitterionic detergents, highlighting the multitude of different detergent interactions with enzymes that target amphiphilic substrates and providing means of trapping a protein in a metastable state. We propose a model for destabilization where monomers via various binding sites on the native state prime it for interacting with micelles in a destabilizing fashion, whereas only micelles halt refolding due to the absence of these monomer-binding sites in the denatured state.

Keywords: Cutinase; Detergent; Phosphocholine; Stability; Activity

Testing the paradigm that the denaturing effect of urea on protein stability is offset by methylamines at the physiological concentration ratio of 2:1 (urea:methylamines) by Laishram Rajendrakumar Singh; Tanveer Ali Dar; Inamul Haque; Farah Anjum; Ali Akbar Moosavi-Movahedi; Faizan Ahmad (pp. 1555-1562).

The intra- and extracellular urea concentration in many organisms is sufficiently high to destabilize (inhibit) many proteins, yet organisms survive and function. The generally accepted explanation is the counteracting hypothesis, which holds that methylamines stabilize proteins and oppose the deleterious effect of urea. The two osmolytes are typically found at 2:1 concentration ratio (urea:methylamine) under physiological conditions. Does this mean that this ratio holds for all proteins in a cell? The present study tests the counteracting hypothesis by determining the effects of urea and methylamines (trimethylamine N-oxide and sarcosine), singly and in combination at a concentration ratio of 2:1 (urea:methylamine) on the thermal denaturation equilibrium, native state↔denatured state of three different proteins (α-lactalbumin, lysozyme and Ribonuclease-A). We show here that the molar concentration of a methylamine required to offset the denaturing effect of urea at a given concentration is different for different proteins.

Keywords: Abbreviations; lzm; hen egg white lysozyme; RNase-A; ribonuclease-A; α-LA; apo-α-lactalbumin; TMAO; trimethylamine N-oxide; T; _m; midpoint of denaturation; Δ; G; _D; denaturational Gibbs energy change; GdmCl; guanidinium chloride; ε; molar absorption coefficient; [; θ; ]; ₂₂₂; mean residue ellipticity at 222 nmProtein stability; Urea-methylamine compensation; Osmolytes; Heat denaturation; Protein folding; Trimethylamine-N-Oxide

Thiocyanate hydrolase, the primary enzyme initiating thiocyanate degradation in the novel obligately chemolithoautotrophic halophilic sulfur-oxidizing bacterium Thiohalophilus thiocyanoxidans by Ekaterina Yu. Bezsudnova; Dimitry Yu. Sorokin; Tamara V. Tikhonova; Vladimir O. Popov (pp. 1563-1570).

Thiohalophilus thiocyanoxidans is a first halophilic sulfur-oxidizing chemolithoautotrophic bacterium capable of growth with thiocyanate as an electron donor at salinity up to 4 M NaCl. The cells, grown with thiocyanate, but not with thiosulfate, contained an enzyme complex hydrolyzing thiocyanate to sulfide and ammonia under anaerobic conditions with carbonyl sulfide as an intermediate. Despite the fact of utilization of the «COS pathway», high cyanase activity was also detected in thiocyanate-induced cells. Three-stage column chromotography resulted in a highly purified thiocyanate-hydrolyzing protein with an apparent molecular mass of 140 kDa that consists of three subunits with masses 17, 19 and 29 kDa. The enzyme is a Co,Fe-containing protein resembling on its function and subunit composition the enzyme thiocyanate hydrolase from the Betaproteobacterium Thiobacillus thioparus. Cyanase, copurified with thiocyanate hydrolase, is a bisubstrate multisubunit enzyme with an apparent subunit molecular mass of 14 kDa. A possible role of cyanase in thiocyanate degradation by T. thiocyanoxidans is discussed.

Keywords: Halophilic sulfur-oxidizing bacteria; Thiohalophilus thiocyanoxidans; Thiocyanate; Carbonyl sulfide; Thiocyanate hydrolase; Cyanase

Effect of a disulfide bond on mevalonate kinase by Xiusheng Chu; Wenhua Yu; Long Wu; Xiaojun Liu; Nan Li; Ding Li (pp. 1571-1581).

Mevalonate kinase is one of ATP-dependent enzymes in the mevalonate pathway and catalyzes the phosphorylation of mevalonate to form mevalonate 5-phosphate. In animal cells, it plays a key role in regulating biosynthesis of cholesterol, while in microorganisms and plants, it is involved in the biosynthesis of isoprenoid derivatives that are one of the largest groups of natural products. Crystal structure and sequence alignment show that a unique disulfide bond exists in mevalonate kinase of thermostable species Methanococcus jannaschii, but not in rat mevalonate kinase. In the present study, we investigated the effect of the disulfide bond in M. jannaschii mevalonate kinase and an engineered disulfide bond in rat mevalonate kinase mutant A141C on the properties of enzymes through characterization of their wild-type and variant enzymes. Our result suggests that the Cys107–Cys281 disulfide bond is important for maintaining the conformation and the thermal activity of M. jannaschii mevalonate kinase. Other interactions could also have contributions. The thiol-titration and fluorescence experiment further indicate that rat mevalonate kinase A141C variant enzyme has a new disulfide bond, which makes the variant protein enhance its thermal activity and resist to urea denaturation.

Keywords: Abbreviations; ATP; Adenosine 5′-triphosphate; NADH; Nicotinamide adenine dinucleotide reduced form; SDS; Sodium dodecylsulfate; PAGE; Polyacrylamide gel electrophoresis; PCR; Polymerase chain reaction; MMK; M. jannaschii; mevalonate kinase; RMK; Rat mevalonate kinase; UV/Vis; Ultraviolet-visible spectroscopyMevalonate kinase; Disulfide bond; Thermal activity; Fluorescence; Mevalonate pathway

Changes of net charge and α-helical content affect the pharmacokinetic properties of human serum albumin by Yasunori Iwao; Mikako Hiraike; Ulrich Kragh-Hansen; Katsumi Mera; Taishi Noguchi; Makoto Anraku; Keiichi Kawai; Toru Maruyama; Masaki Otagiri (pp. 1582-1590).

The pharmacokinetics of 17 genetic variants of human serum albumin with single-residue mutations and their corresponding normal albumin were studied in mice. In all cases, the plasma half-life was affected, but only variants with +2 changes in charge prolonged it, whereas changes in hydrophobicity decreased it. Good positive and negative correlations were found between changes in α-helical content taking place in domains I+III and domain II, respectively, and changes in half-lives. No correlation was found to type of mutation or to changes in heat stability as represented by Δ H_v. Liver and kidney uptake clearances were also modified: α-helical changes of domains I+III showed good negative correlations to both types of clearances, whereas changes in domain II only had a good positive correlation to kidney uptake clearance. No correlation between the other molecular changes and organ uptakes was observed. The relatively few correlations between changes in molecular characteristics and the organ uptakes of the variants are most probably due to different handling by plasma enzyme(s) and the various types of cell endocytosis. Of the latter, most lead to destruction of albumin, but at least one results in recycling of the protein. The present information should be useful when designing recombinant, therapeutical albumins or albumin products with a modified plasma half-life.

Keywords: Abbreviations; HSA; human serum albumin; Alb; albumin; Alb A; normal (wild-type) albumin; Δ; H; _v; van't Hoff enthalpyHuman serum albumin; Genetic variant; Pharmacokinetics; Half-life; Hepatic uptake; Renal disposition

Reversible and irreversible unfolding of multi-domain proteins by K.H. Strucksberg; T. Rosenkranz; J. Fitter (pp. 1591-1603).

In contrast to single-domain proteins unfolding of larger multi-domain proteins is often irreversible. In a comparative case study on three different multi-domain proteins (phosphoglycerate kinase: PGK and two homologous α-amylases: TAKA and BLA) we investigated properties of unfolded states and their ability to fold back into the native state. For this purpose guanidine hydrochloride, alkaline pH, and thermal unfolded states were characterized. Structural alterations upon unfolding and refolding transitions were monitored using fluorescence and CD spectroscopy. Static and dynamic light scattering was employed to follow aggregation processes. Furthermore, proper refolding was also investigated by enzyme activity measurements. While for PGK at least partial reversible unfolding transitions were observed in most cases, we found reversible unfolding for TAKA in the case of alkaline pH and GndHCl induced unfolding. BLA exhibits reversible unfolding only under conditions with high concentrations of protecting osmolytes (glycerol), indicating that aggregation of the unfolded state is the main obstacle to achieve proper refolding for this protein. Structural properties, such as number and size of domains, secondary structure contents and compositions within domains, and domain topology were analyzed and considered in the interpretation of differences in refolding behavior of the investigated proteins.

Keywords: alpha-amylase; Aggregation; Unfolded states; Protecting osmolyte; Hydrodynamic radiusAbbreviations; DLS; dynamic light scattering; SLS; static light scattering; CD; circular dichroism; PGK; phosphoglycerate kinase from Bakers yeast; BLA; Bac. licheniformis; α-amylase; TAKA; Aspergillus oryzae; α-amylase; DTT; dithiothreitol; Mops; 3-(N-morpholino)propanesulfonic acid; GndHCl; guanidine hydrochloride; EDTA; ethylenediamine tetraacid; RMSD; Root mean square deviation

Solid-state NMR study and assignments of the KcsA potassium ion channel of S. lividans by Krisztina Varga; Lin Tian; Ann E. McDermott (pp. 1604-1613).

The extraordinary efficiency and selectivity of potassium channels have made them ideal systems for biophysical and functional studies of ion conduction. We carried out solid-state NMR studies of the selectivity filter region of the protein. Partial site-specific assignments of the NMR signals were obtained based on high field multidimensional solid-state NMR spectra of uniformly¹³C,¹⁵N enriched KcsA potassium channel from Streptomyces lividans. Both backbone and sidechain atoms were assigned for residues V76-D80 and P83-L90, in and near the selectivity filter region of the protein; this region exhibits good dispersion and useful chemical shift fingerprints. This study will enable structure, dynamic and mechanistic studies of ion conduction by NMR.

Keywords: Solid-state NMR; Potassium channel; Membrane protein

Thermal denaturation of CP43 studied by Fourier transform-infrared spectroscopy and terahertz time-domain spectroscopy by Yuangang Qu; Hua Chen; Xiaochun Qin; Liangbi Li; Li Wang; Tingyun Kuang (pp. 1614-1618).

Thermal denaturation of CP43 was studied by Fourier transform-infrared (FT-IR) spectroscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and terahertz time-domain spectroscopy (THz-TDS). Under heat treatment, the secondary structure of CP43 changed, and the main thermal transition occurred at 59 °C. During the process, CP43 aggregated at first, and then with increasing temperature degraded. The low-frequency collective vibrational modes of CP43 changed with increasing temperature and decreasing mass. THz-TDS is a new technique used to study the conformational state of a molecule, and it is the first use of this technique to study the photosynthesis membrane proteins in this paper. The results presented here demonstrate that THz-TDS has both advantages and disadvantages in monitoring the thermal denaturation of membrane proteins, which is important in applying THz-TDS technique to study of biomolecules.

Keywords: Abbreviations; β-Car; β-carotene; CD; circular dichroism; Chl; chlorophyll; DM; β-dodecyl maltoside; FIR; far-infrared; FT-IR; Fourier transform-infrared; IR; infrared; LHCII; light-harvesting complex; PS II; photosystem II; RC; reaction center; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; THz-TDS; terahertz time-domain spectroscopyCP43; Denaturation; Fourier transform-infrared spectroscopy; Heat treatment; Terahertz time-domain spectroscopy

Promotion of insulin aggregation by protein disulfide isomerase by Ryosuke Maeda; Kazuyoshi Ado; Naohiro Takeda; Yoshihiro Taniguchi (pp. 1619-1627).

We examined the aggregation of insulin as a result of reduction of disulfide bonds catalyzed by protein disulfide isomerase (PDI) using various techniques. We demonstrated the kinetic correlation between PDI-catalyzed insulin reduction and the aggregate formation, the relationship between aggregation and amyloid formation, and the structural information on the secondary structure of the aggregates. The initial rate of PDI-catalyzed reduction of insulin, apparent rate constants of aggregate growth for sigmoidal features, and lag times were obtained by changing the PDI concentration, temperature, and insulin concentration. In situ kinetics were studied using the dyes; thioflavin T (ThT) and Congo red (CR) in addition to turbidimetry with the insulin reduction by PDI. The ThT and CR binding assay revealed sigmoidal kinetics, and the spectrum of binding CR showed a red shift against time, suggesting an orderly formation of insulin aggregates. The secondary structure of the PDI-promoted insulin aggregates showed antiparallel β-sheet conformation by FT-IR measurement.

Keywords: Abbreviations; PDI; protein disulfide isomerase; GR; glutathione reductase; GSH; glutathione reduced form; GSSG; glutathione oxidized form; NADPH; β-nicotinamide adenine dinucleotide phosphate; ThT; thioflavin T; CR; Congo red; FT-IR; Fourier transform infraredProtein disulfide isomerase; Insulin; Disulfide bond; Protein aggregation; Kinetic analysis; Amyloid

Mutation of Tyr375 to Lys375 allows medium-chain acyl-CoA dehydrogenase to acquire acyl-CoA oxidase activity by Jia Zeng; Yuandong Liu; Long Wu; Ding Li (pp. 1628-1634).

Medium-chain acyl-CoA dehydrogenase (MCAD) and acyl-CoA oxidase (ACO) are key enzymes catalyzing the rate-determining step for the β-oxidation of fatty acids. Tyr375 of MCAD is conserved in all acyl-CoA dehydrogenases and is an important residue for substrate binding. Four Tyr375 variant enzymes of rat liver MCAD were obtained through site-directed mutagenesis. Y375K was found to have intrinsic acyl-CoA oxidase activity, which was confirmed using HPLC analysis, while the wild-type and other Tyr375 variant enzymes did not show detectable oxidase activity. The kinetic parameters for the oxidase activity of Y375K variant enzyme were determined to be k_cat of 320±80 h−¹ and K_M of 30±15 μM using hexanoyl-CoA as the substrate. The oxidase activity of Y375K increased more than 200 times compared with that reported for the MCAD wild-type enzyme from mammalian sources. Molecular modeling study shows that the solvent accessible area for Y375K variant enzyme is wider than that of the wild-type enzyme, which indicates that Tyr375 may function as a switch against solvent accession. The mutation of this residue to Lys375 allows molecular oxygen to enter into the catalytic site serving as the electron acceptor for the reduced FAD cofactor.

Keywords: Abbreviations; ACD; acyl-CoA dehydrogenase; ACO; acyl-CoA oxidase; CD; circular dichroism; DTT; dithiothreitol; FAD; flavin adenine dinucleotide; iBD; isobutyryl-CoA dehydrogenase; IPTG; isopropyl-β-; d; -thiogalactopyranoside; MCAD; medium-chain acyl-CoA dehydrogenase; PAGE; polyacrylamide gel electrophoresis; PCR; polymerase chain reaction; SAR; solvent accessible area; SCAD; short chain acyl-CoA dehydrogenase; SDS; sodium dodecylsulfate; UV/Vis; ultraviolet-visible spectroscopyAcyl-CoA dehydrogenase; Acyl-CoA oxidase; β-Oxidation; Fatty acid; Solvent accessible area

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BBA - Proteins and Proteomics (v.1774, #12)