Biochemical characterization of a variant human medium-chain acyl-CoA dehydrogenase with a disease-associated mutation localized in the active site.

Medium-chain acyl-CoA dehydrogenase (MCADH) deficiency, an autosomal recessive inherited disorder, is the most common genetic disorder in mitochondrial beta-oxidation in humans. In addition to one prevalent disease-causing mutation (K304E), a series of rarer mutations has been reported, but none of these has yet been characterized in detail. We report here on the biochemical characterization of the purified recombinant mutant protein in which threonine is replaced by alanine at position 168 of the mature protein (T168A-MCADH). It is the first mutation to be found in patients that is located in the active site of the enzyme. Thr-168 is hydrogen-bonded to the flavin N(5) of the cofactor FAD. The thermostability of T168A-MCADH is markedly decreased compared with human wild-type MCADH (hwt-MCADH). Catalytic activity with ferricenium as acceptor is lowered by 80% and with the natural acceptor electron-transferring flavoprotein by over 90% compared with hwt-MCADH. In the mutant the extent of flavin semiquinone formed on reduction is approx. 50% that of hwt-MCADH. The pK reflected by the pH-dependence of Vmax is shifted from approx. 8.2 (hwt-MCADH) to approx. 7 (T168A-MCADH) and the rates of enzyme flavin reduction (stopped-flow measurements) are only approx. 1/10 those of the parent enzyme. These properties are discussed in the light of the possible mechanisms leading to disease in humans.

Biochemical characterization of purified, human recombinant Lys304-->Glu medium-chain acyl-CoA dehydrogenase containing the common disease-causing mutation and comparison with the normal enzyme.

Recombinant, normal human medium-chain acyl-CoA dehydrogenase (MCADH) and the common, human disease-causing K304E mutant ([Glu304]MCADH) protein were expressed in Escherichia coli using an optimized system, and the enzymes were purified to apparent homogeneity. The crucial factor leading to the production of active [Glu304]MCADH protein is the expression in E. coli cells at reduced temperature (28 degrees C). Expression in the same system at 37 degrees C results in very low amounts of active mutant protein. Several catalytic and physicochemical parameters of these two proteins have been determined and were compared to those of purified pig kidney MCADH. Although [Glu304]MCADH has approximately the same rate of substrate reduction with dodecanoyl-CoA and the same V(max) as human MCADH with the best substrate for the latter, octanoyl-CoA, the K(m) in the mutant MCADH is fourfold higher, which generates a correspondingly lower catalytic efficiency. Importantly, V(max) obtained using the natural acceptor, electron transfer flavoprotein, is only a third that for human MCADH. The V(max)/K(m) versus chain-length profile of the mutant shows a maximum with dodecanoyl-CoA which differs markedly from that of human MCADH, which has maximal efficiency with octanoyl-CoA. The substrate specificity of the mutant is broader with a less pronounced activity peak resembling long-chain acyl-CoA dehydrogenase. The purified mutant enzyme exhibits a reduced thermal stability compared to human wild-type MCADH. The major difference between the two proteins expressed in E. coli is the more pronounced lability of the K304E mutant in crude extracts, which suggests a higher susceptibility to attack by endogenous proteases. Differences between tetrameric [Glu304]MCADH which survives the first step(s) of purification and corresponding MCADH are minor. The overall differences in properties of [Glu304]MCADH together with its impaired folding and tetramer assembly may contribute to the generation of the abnormalities observed in patients homozygous for the K304E mutation.

Medium-long-chain chimeric human Acyl-CoA dehydrogenase: medium-chain enzyme with the active center base arrangement of long-chain Acyl-CoA dehydrogenase.

The catalytically essential glutamate residue that initiates catalysis by abstracting the substrate alpha-hydrogen as H+ is located at position 376 (mature MCADH numbering) on loop JK in medium chain acyl-CoA dehydrogenase (MCADH). In long chain acyl-CoA dehydrogenase (LCADH) and isovaleryl-CoA dehydrogenase (IVDH), the corresponding Glu carrying out the same function is placed at position 255 on the adjacent helix G. These glutamates thus act on substrate approaching from two opposite regions at the active center. We have implemented the topology of LCADH in MCADH by carrying out the two mutations Glu376Gly and Thr255Glu. The resulting chimeric enzyme, "medium-/long" chain acyl-CoA dehydrogenase (MLCADH) has approximately 20% of the activity of MCADH and approximately 25% that of LCADH with its best substrates octanoyl-CoA and dodecanoyl-CoA, respectively. MLCADH exhibits an enhanced rate of reoxidation with oxygen, however, with a much narrower substrate chain length specificity that peaks with dodecanoyl-CoA. This is the same maximum as that of LCADH and is thus significantly shifted from that of native MCADH (hexanoyl/octanoyl-CoA). The putative, common ancestor of LCADH and IVDH has two Glu residues, one each at positions 255 and 376. The corresponding MCADH mutant, Thr255Glu (glu/glu-MCADH), is as active as MCADH with octanoyl-CoA; its activity/chain length profile is, however, much narrower. The topology of the Glu as H+ abstracting base seems an important factor in determining chain length specificity and reactivity in acyl-CoA dehydrogenases. The mechanisms underlying these effects are discussed in view of the three-dimensional structure of MLCADH, which is presented in the accompanying paper [Lee et al. (1996) Biochemistry 35, 12412-12420].

Purification and characterization of a novel acid-soluble nuclear protein from developing embryos of the camel tick Hyalomma dromedarii (Acarina: Ixodidae).

A novel acid-soluble protein has been extracted from nuclei of developing embryos of H. dromedarii ticks and purified to homogeneity. This tick embryo basic protein (TEBP) was predominant during the cleavage stage of tick embryogenesis, whereas the complete set of histones was detectable at the late cleavage stage. The amount of TEBP reaches a maximum value at day 9 after oviposition. Thereafter, the original N-terminal dipeptide (leucine-serine) is eliminated. This coincides with the start of organogenesis. In spite of its low molecular mass, TEBP seems to be related to histone H1 in some properties such as solubility in perchloric acid and binding affinity to DNA. A task for the future will be to define the role of this protein as a counterpart of the histones for the genome organization during embryogenesis.

Convergent evolution of amino acid usage in archaebacterial and eubacterial lineages adapted to high salt.

Chemical composition and physical properties of the total protein of Haloferax mediterranei, a halophilic archaebacterium requiring high salt concentration for growth, of Halomonas elongata, a halotolerant eubacterium able to grow at any concentration of salt, and of Escherichia coli B, a eubacterium related to H. elongata, unable to grow at high salt concentration, were compared using robust standard biochemical methods. The distribution of amino acid abundancies in the bulk protein from H. elongata was found to be intermediate between that from H. mediterranei and that from E. coli. The two high-salt-adapted organisms displayed an enrichment in aspartic acid and glutamic acid together with an impoverishment in lysine as compared to E. coli. This signature in amino acid usage is reflected in the charge distribution of proteins, as revealed by anion exchange chromatography of crude cell extracts. Since H. elongata diverged from H. mediterranei more than three billion years ago, the resemblance of their amino acid usages can be interpreted as a convergent imprint of their common habitats onto the chemical constitution of their proteins.

The role of the adsorption complex in the termination of filamentous phage assembly.

The adsorption complex of filamentous phage fd consists of two minor coat proteins, g3p and g6p, and is considered to be not only a structural entity, but also a functional unit to terminate phage assembly. Cells were infected with phage M13am8H1, which cannot assemble because it lacks the major coat protein g8p, although producing all of the other minor coat proteins. The membranes of infected cells were solubilized and analysed by non-denaturing PAGE and gel filtration. The data suggest the presence of the adsorption complex in these membranes. Furthermore, the non-polar gene 3 amber-mutant phage R171 was shown to lack g6p in the phage coat as well. The termination of assembly of this phage is disturbed, resulting in synthesis of polyphages. Electron micrographs and transient electrical birefringence show that these polyphages are eight times longer as compared to unit length phage. From these results, we conclude that the formation of the g3p-g6p complex is essential for correct termination of filamentous phage assembly.

The adsorption protein of filamentous phage fd: assignment of its disulfide bridges and identification of the domain incorporated in the coat.

The mature adsorption protein (g3p) of filamentous phage fd consists of 406 amino acids. It contains eight cysteine residues in total. To determine the disulfide bond pattern, purified g3p was proteolytically digested, and the resulting peptides were separated using RP-HPLC. N-terminal sequencing and mass spectrometry of cysteine-containing fragments showed that each cysteine is involved in an intramolecular disulfide bond. The cystine sites are Cys7-Cys36, Cys46-Cys53, Cys188-Cys201, and Cys354-Cys371. In the native conformation of g3p, none of the disulfide bridges is accessible to alkylating agents after treatment with DTT. The cystine sites seem therefore to be located in the interior of the folded molecule or are shielded by components of the phage envelope. The part of g3p which is incorporated in the phage coat and hence is inaccessible to proteolytic cleavage was identified after treatment of phage particles with subtilisin: Immunoblots performed with antibody directed against g3p revealed essentially one g3p fragment with an apparent molecular weight of approximately 17,000 which resisted the proteolytic attack. The amino terminus of this peptide was determined to be glycine 254. This amino acid position correlates with the carboxy-terminal end of the second glycine-rich region (amino acids 218-256) in the primary structure of g3p. The notion that the extended carboxy-terminal g3p fragment is indeed entirely buried within the phage envelope is further supported by the fact that only polyclonal antibodies raised against purified g3p are able to react with this peptide, whereas those against phage coat-associated g3p are not.

Novel inactive and distinctively glycosylated forms of butyrylcholinesterase from chicken serum.

Three different homologues of butyrylcholinesterase (BChE) with 75-, 62-, and 54-kDa subunit size are isolated from adult chicken serum, and all show very low or zero enzyme activity. Although the active BChE from serum with a subunit size of 81 kDa forms tetramers, the 75-kDa protein is isolated as a dimer. The homology of the 75-kDa protein with active BChE is shown by immunoreactivity with BChE-specific monoclonal antibodies, by coisolation with the active BChE, and by their identical first six N-terminal amino acids. By deglycosylation of these proteins and by their differential lectin binding, we show that the active BChE is an N-glycosylated protein of the triantennary type, whereas the inactive 75-kDa protein is O-glycosylated. These data show for the first time the existence of (1) multiple inactive forms of BChE, (2) secreted inactive cholinesterases, because they are found in serum, and (3) an O-glycosylated cholinesterase. Because cholinesterases can regulate neurite growth in vitro by a nonenzymatic mechanism, these data strongly support that both inactive and active forms of BChE may be involved in noncholinergic communication, possibly depending on particular glycosylation patterns.

The adsorption protein of bacteriophage fd and its neighbour minor coat protein build a structural entity.

The adsorption protein g3p and another minor coat protein, g6p, are located at one end of the filamentous bacteriophage fd [Grant, R.A., Lin, T.C., Konigsberg, W.E. & Webster, R.E. (1981) J. Biol. Chem. 256, 539-546]. Both proteins, representing the proximal tip, were detached as an entity by a technique that allowed for gentle solubilization. Disrupting the phage particle with the detergent sodium deoxycholate and chloroform dissociates the major coat protein g8p, frees the phage DNA, but leaves g3p and g6p associated with each other. The g3p-g6p complex, which we termed the adsorption complex, and an oligomeric form of g3p with lower molecular mass were isolated and purified by gel-filtration chromatography in the presence of deoxycholate. These different oligomeric structures of g3p showed a different mobility in non-denaturing polyacrylamide-gel electrophoresis. Both forms were also found in non-denaturing polyacrylamide-gel electrophoresis from deoxycholate- and Triton-X-100-solubilized phage without prior chromatographic separation. The two oligomeric forms of g3p are composed of two g3p polypeptide chains in the case of the low-molecular-mass species, and four g3p and four g6p polypeptide chains for the adsorption complex.

Characterization of wild-type human medium-chain acyl-CoA dehydrogenase (MCAD) and mutant enzymes present in MCAD-deficient patients by two-dimensional gel electrophoresis: evidence for post-translational modification of the enzyme.

Two-dimensional gel electrophoresis was used to study and compare wild-type medium-chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3) and mis-sense mutant enzyme found in patients with MCAD deficiency. By comparing the patterns for wild-type and mutant MCAD expressed in Escherichia coli or in eukaryotic COS-7 cells we demonstrate that variants with point mutations changing the net charge of the protein can be readily resolved from the wild-type protein. After expression of the cDNA in eukaryotic cells two spots representing mature MCAD can be distinguished, one with an isoelectric point (pI) corresponding to that obtained for the mature protein expressed in E. coli and another one shifted to lower pI. This demonstrates that MCAD protein is partially modified after transport into the mitochondria and removal of the transit peptide. The observed pI shift would be compatible with phosphorylation of one aspartic acid residue per monomer. Comparison of pulse labeling and steady-state amounts of MCAD protein in overexpressing COS-7 cells confirms that K304E MCAD is synthesized and transported into mitochondria in amounts similar to the wild-type protein, but is degraded much more readily. For wild-type MCAD, the spot representing the nonmodified form predominates after pulse labeling while that representing the modified form is relatively stronger in steady state, demonstrating that the modification occurs in mitochondria after the transit peptide has been removed. For K304E mutant MCAD, the nonmodified spot is relatively stronger both in pulse labeling and in steady state, indicating that either the efficiency of modification or the stability of the modified form is affected by the K304E mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural characterization and biological activity of recombinant human epidermal growth factor proteins with different N-terminal sequences.

The primary structures and molecular homogeneity of recombinant human epidermal growth factors from different suppliers were characterized and their biological activities evaluated by a standard DNA synthesis assay. Molecular weight determinations using 252Cf-plasma-desorption and electrospray mass spectrometry in combination with N- and C-terminal sequence analysis and determination of intramolecular disulfide bridges revealed that one recombinant protein had the correct human-identical structure (54 aa residues; 6347 Da). In contrast, a second recombinant protein (7020 Da) was found to contain a pentapeptide (KKYPR) insert following its N-terminal methionine. This structural variant showed a significant reduction in its capacity to stimulate DNA synthesis.

Interchangeability of the adsorption proteins of bacteriophages Ff and IKe.

The wild-type adsorption protein (g3p) of filamentous phage IKe cannot be exchanged with its analogous protein in the related Ff (M13, fd, and f1) phage particles. Deletion mutants of the protein, however, are assembled into Ff phage particles. These hybrid Ff phage particles bearing deleted IKe g3p attach to N pili, thus conserving the host attachment property of the protein but not its infection-initiating function. This means that the attachment specificity is determined by IKe g3p independently of other phage components in contact with it. Infection initiation function, the process in which phage DNA is released into the host, in contrast seems to require either more complex structural features of the protein (for example, a certain oligomeric structure) provided only in the original particle, or a concerted action of g3p with another particle component, not replaceable by its homologous counterpart in the related phage.

Subtilisin removes the surface layer of the phage fd coat.

The major coat protein of native filamentous phage fd is vulnerable to digestion by subtilisin, but not by any of a number of other proteolytic enzymes. Degradation by the non-specific protease subtilisin occurs at specific sites in the N-terminal portion of g8p. The N-terminal part of the protein is considered to be the outer layer of a two-layered coat. Thus, subtilisin treatment results in a monolayered phage particle. These particles possess the morphology and stability of native phage fd. Furthermore, subtilisin proteolysis proved to be an efficient instrument in detecting variations in the topology of the g8p of related filamentous phages.

The adsorption protein of phage IKe. Localization by deletion mutagenesis of domains involved in infectivity.

We constructed a set of plasmid-encoded internal deletion mutants within the gene for the adsorption protein (g3p) of phage IKe. All mutant proteins still contain the signal and membrane anchor sequence, as those are known to be indispensable for proper localization and hence assembly of the g3p into phage. These various deletions comprise all internal parts of the protein and are properly incorporated into phage, which remarkably shows that signal and anchor sequence are sufficient for incorporation of g3p. The data furthermore reveal that two separate sections within the IKe g3p are essential for infection: one amino-terminal, preceding the glycine-rich stretch, and the other carboxy-terminal. We conclude that this latter domain is involved in penetration because mutants lacking it are not infectious, but still bind to the receptor. The amino-terminal region, essential for infection, bears the receptor-recognizing domain and a sequence homologous to the penetration domain of the evolutionary related Ff phages, which is probably also involved in penetration of phage IKe. The prominent glycine-rich stretch of the IKe g3p is not essential for infection but significantly promotes it.

Release of periplasmic proteins induced in E. coli by expression of an N-terminal proximal segment of the phage fd gene 3 protein.

We used the enzymes beta-lactamase and alkaline phosphatase to quantitatively evaluate the release of periplasmic proteins from E. coli cells transformed by plasmids harboring gene 3 of phage fd. Different deletion mutants of gene 3 released varying fractions of the enzymes. From these results we conclude that essentially the amino-terminal proximal part, upstream of the first glycine-rich region but not this region itself, is responsible for the excretion of periplasmic proteins in E. coli cells expressing the gene 3 protein of phage fd.

Genetic analysis of the transfer region of the IncN plasmid N3.

Using lambda::Tn5 insertion mutagenesis and screening for conjugation, the boundaries of the IncN plasmid N3 transfer region were determined. Sensitivity to phage IKe infection was used to monitor that part of the N3 transfer region which harbours genes for pilus synthesis and assembly. We cloned this region, creating plasmid pBG21. Escherichia coli cells transformed with pBG21 became sensitive to phage IKe and produced pili, as shown by electron microscopy. Various plasmid constructions containing parts of the pilus-encoding region were used for expression in a minicell system and for expression in an in vitro translation system, thus characterizing for the first time some of the gene products of domain I (Winans and Walker, 1985a) of the transfer region.

Characterization of wild-type and an active site mutant of human medium chain acyl-CoA dehydrogenase after expression in Escherichia coli.

The cDNA of human medium chain acyl-CoA dehydrogenase (MCADH) was modified by in vitro mutagenesis, and the sequence encoding the mature form of MCADH was introduced into an inducible expression plasmid. We observed synthesis of the protein in Escherichia coli cells transformed with this plasmid with measurable MCADH enzyme activity in cell extracts. Glutamic acid 376, which has been proposed by Powell and Thorpe (Powell, P. J., and Thorpe, J. (1988) Biochemistry 27, 8022-8028) as an essential residue and the proton-abstracting base at the active site of the enzyme, was mutated to glutamine. After expression in bacteria of this plasmid, the corresponding extracts show no detectable MCADH activity, although mutant MCADH-protein production was detected by protein immunoblots. The mature enzyme and the Gln376 mutant were purified to apparent homogeneity. The wild-type enzyme is a yellow protein due to the content of stoichiometric FAD and had a specific activity which is 50% of MCADH purified from pig kidney. The Gln376 mutant is devoid of activity (less than 0.02% that of wild type, expressed enzyme) and is green because of bound CoA persulfide. Properties of the mutant enzyme suggest that the Glu376----Gln change specifically affects substrate binding. These results prove that Glu376 plays an important role in the initial step of dehydrogenation catalysis.

Dissection of functional domains in phage fd adsorption protein. Discrimination between attachment and penetration sites.

We constructed a set of deletion mutants in the attachment protein of phage fd. These mutants lack sequences coding for sections in the amino-terminal half. All the mutants that comprise a leader sequence are incorporated into phage particles. Our data strongly suggest a bipartite organization of the amino-terminal domain with (1) a region for receptor recognition and (2) a region that is necessary for penetration of the DNA into the host cell. These regions were mapped. Some evidence suggesting different roles for gene 3 protein in penetration of the outer and inner membrane are discussed. We demonstrate that the phenotypes caused by gene 3 protein in host cells can be subdivided into two groups with different sequence requirements: (1) phenotypes related to outer membrane disturbance; and (2) phenotypes related to the tolQRA transport system.