Refolding and purification of Cephalosporium acremonium deacetoxycephalosporin C synthetase/hydroxylase from granules of recombinant Escherichia coli.

The gene for bifunctional deacetoxycephalosporin C synthetase/hydroxylase of Cephalosporium acremonium was cloned and overexpressed as an insoluble and inactive enzyme in granules of recombinant Escherichia coli. About 40-60% of expected synthetase activity along with 50-80% protein purity could be recovered directly from granular extracts with only a single empirically optimized refolding step. Further purification to homogeneity was achieved by a single anion-exchange-chromatographic step in the presence of denaturing concentrations of urea. The main obstacle to converting the homogeneous unfolded protein into the active enzyme was a urea-dependent aggregation during refolding that led to irreversible enzyme inactivation. Information obtained from refolding studies using gel-filtration HPLC, fluorescence spectroscopy and disulphide analysis led to an optimal enzyme refolding scheme that resulted in a highly active (i.e. 65-75% of the expected activity) and moderately stable fungal synthetase/hydroxylase.

Purification and properties of NADPH-dependent tylosin reductase from Streptomyces fradiae.

A reductase of Streptomyces fradiae was speculated to catalyze reduction of tylosin to relomycin, an industrially undesirable product. The activity of tylosin reductase was closely related to bacterial growth, suggesting involvement of the enzyme in a primary metabolism. The reductase activity was improved significantly in vivo and in vitro. The enzyme was also partially stabilized in vitro. Using a simple five-step chromatographic procedure, the reductase was purified 480-fold to apparent homogeneity. The purified reductase had a molecular mass of 270 kDa and consisted of two different subunits of 26 and 7 kDa at 1:1 ratio. The enzyme exhibited an absorption maximum at 405 nm and was inhibited by exogenous FAD or FMN, indicating a flavin as its prosthetic group. Tylosin reductase was optimally active at pH 7.0-7.2 and 40 degrees C with NADPH as a preferred electron donor. The Km of the enzyme for tylosin was 1.4 mM and that for NADPH was 0.15 mM. The Vmax for the enzymatic reaction was 917 mumol of tylosin formed/min/mg protein. The enzymatic conversion of tylosin to relomycin was coupled to that of NADPH to NADP+ at a stoichiometric ratio of 1:1. Tylosin reductase showed a broad substrate specificity toward all macrolide aldehydes (as normal and shunt metabolites of tylosin biosynthesis) tested. Thus, the enzyme may have a physiological role of macrolide detoxification for the bacterium.

The functional role of cysteines in isopenicillin N synthase. Correlation of cysteine reactivities toward sulfhydryl reagents with kinetic properties of cysteine mutants.

Isopenicillin N synthase (IPNS) from Cephalosporium acremonium contains 2 cysteine residues in positions 106 and 255 which are invariant in all IPNS sequences reported to date (Miller, J.R., and Ingolia, T.D. (1989) Mol. Microbiol. 3, 689-695). Although these residues have been postulated to play a role in catalysis (Samson, S.M., Chapman, J.L., Belagaje, R., Queener, S., and Ingolia, T.D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5705-5709) as well as enzyme inactivation (Perry, D., Abraham, E.P., and Baldwin, J.E. (1988) Biochem. J. 255, 345-351) little information exists regarding their oxidation state and reactivity. In this paper, the functions of these cysteines have been addressed by chemical modification techniques in combination with site-directed mutagenesis. In the intact wild type protein, both cysteines are inert toward 5,5'-dithiobis-(2-nitrobenzoic acid) and iodoacetic acid. However, Cys-106, but not Cys-255, can be slowly modified by N-ethylmaleimide, and its modification is partially blocked by the presence of a substrate analog inhibitor. Complete modification of both cysteines by sulfhydryl reagents requires unfolding of the protein but not the presence of a disulfide reducing agent. The thiol content of IPNS is shown to be the same before and after exposing the enzyme to substrate even though during catalysis the enzyme is rapidly inactivated. The impact on catalysis due to alteration of the cysteines has been assessed using three site-specific mutants: Cys-106----Ser, Cys-255----Ser, and Cys-106,255----Ser. These mutant proteins have been purified as apoenzymes with the nature of the mutation verified by peptide mapping. The stoichiometry of metal required for activity remains as one equivalent of Fe2+/mol of enzyme in the mutants as in wild type IPNS. Compared with wild type, Cys-255----Ser shows a reduction in Vmax by 33%, and an increase in Km by 1.4-fold, while Cys-106----Ser and Cys-106,255----Ser, which have identical kinetic properties, exhibit a decrease in Vmax by 63% but an elevation of Km by 14-fold. The data presented demonstrate that 1) both cysteines are free thiols; 2) Cys-106 is more exposed than Cys-255; 3) substrate-induced inactivation is not caused by cysteine modification; 4) neither cysteine is absolutely essential for bond making or breaking events during catalysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthetic peptides derived from the nonmuscle myosin light chains are highly specific substrates for protein kinase C.

The phosphorylation of synthetic peptides derived from the NH2-terminal sequence of smooth-muscle myosin was studied with purified protein kinase C. The protein kinase C phosphorylation domain included both serine residues and threonine residues in the sequence SSKRAKAKTTKKR(G), denoted myosin light chain (1-13) (MLC(1-13)). Kinetic analysis of MLC(1-13) and truncated peptides derived from the parent peptide established that removal of the serine residues had little effect on protein kinase C reactivity. MLC(1-13) had a V/K of 2.4 min-1.mg-1, whereas the V/K of MLC(3-13) was 3.0 min-1.mg-1. Removal of Lys-3 resulted in a 50% decrease in V/K which was attributable to a 50% decrease in apparent Vmax.Arg-4 was established as a significant protein kinase C specificity determinant, since the apparent Km increased 7-fold and the Vmax decreased 3-fold when the parent peptide was truncated at that residue. All peptides studied required calcium and lipid effectors for full activity with protein kinase C, indicating that they are Class C substrates as defined by Bazzi and Nelsestuen (Biochemistry 26 (1987) 5002) for protein kinase C. Other protein kinases, including cyclic AMP- and cyclic GMP-dependent protein kinase, S6/H4 kinase, myosin light-chain kinase and calcium/calmodulin-dependent kinase II, had little or no activity with these peptides. In studies on the purification of lymphosarcoma protein kinase C by several chromatographic procedures, the results showed that the myosin light-chain peptides can provide convenient and well-characterized substrates for purification and mechanistic studies of protein kinase C biochemistry.

Structural analysis of the calcium-binding protein calmodulin from Ascaris suum obliquely striated muscle.

Calmodulin was purified from the obliquely striated skeletal muscle of Ascaris suum. The calmodulin had a molecular weight of 16,400 and the amino acid composition indicated it is highly similar to other purified calmodulins, showing insignificant variation in 12 of 17 residues. In the residues that showed variation, a trend towards conservative substitution was observed. Spectrophotometric absorption maxima of 276 nm and 283 nm were observed. A molar absorption coefficient of 7,800 was calculated. Calcium-dependent binding to phenothiazine Affi-Gel confirmed that calcium binding induces conformation changes characteristic of calmodulin. Double reciprocal analysis of phosphodiesterase activation by A. suum calmodulin gave a Kapp of 40 nM.

Nonmuscle myosin phosphorylation sites for calcium-dependent and calcium-independent protein kinases.

Thymus myosin, light chains and a synthetic peptide (S-S-K-R-A-K-A-K-T-T-K-K-R-P-Q-R-A-T-S-N-V-F-S) corresponding to the N-terminal sequence of smooth muscle myosin light chains were compared as substrates for calcium/calmodulin-dependent protein kinase (MLCK), calcium/phospholipid-dependent protein kinase (PKC), and a MgATP-activated protein kinase (H4PK) from lymphoid cells. All protein kinases catalyzed phosphorylation of the substrates although H4PK showed higher affinity for isolated light chains and the peptide. Phosphoamino acid analysis and analysis of thermolysin peptides established that PKC catalyzed phosphorylation of threonine-9 or 10. In addition, PKC and H4PK catalyzed phosphorylation at serine-19, the MLCK site. Collectively the data support the hypothesis that myosin filament assembly in nonmuscle cells may be regulated by a variety of calcium-dependent and calcium-independent protein kinases.