Brachyurins--serine collagenolytic enzymes from crabs

The properties of brachyurins, proteolytic enzymes belonging to a new subfamily of chymotrypsin-like proteases, are considered. These enzymes, found in various species of crustacean, exhibit mixed substrate specificity and a marked collagenolytic activity. The enzymatic and physicochemical properties of brachyurins I and their primary and spatial structures are discussed in detail. A separate chapter is devoted to the preparations of collagenases from the hepatopancreas of king crab: their action on the damaged skin and use in medicine.     

Cold-adapted and mesophilic brachyurins

Two different types of brachyurins, termed I and II, have been described in the literature. Within type I there are two subtypes, Ia and Ib. The prototype for the type I brachyurins is Fiddler crab collagenase I. Its cold-adapted analogue from Antarctic krill, termed euphaulysin, shares many of its characteristics. Both enzymes are distinguished by their broad substrate specificity as well as the ability to cleave collagen. The precursor form of euphaulysin has been expressed in Pichia pastoris and processed to its fully active form using cod trypsin. A molecular model of euphaulysin, based on the known crystal structure of crab collagenase I, indicates that the core structure of these enzymes is almost identical. As a cold-adapted enzyme, euphaulysin has a higher catalytic efficiency than crab collagenase I. It is also more sensitive to thermal inactivation and autolysis. Furthermore, euphaulysin has an increased length of several surface loops compared to crab collagenase I. Extended surface loops have been suggested to play a role in the cold activity of some bacterial enzymes. Sensitivity to autolysis is an important factor which contributes to the thermal instability of euphaulysin. Substitution of a highly exposed residue in the 'autolysis loop' of euphaulysin resulted in an increased stability of the enzyme towards thermal inactivation without altering its catalytic efficiency.     

Structural and kinetic characterization of native laccases from Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii

A comparative study has been performed on five native laccases purified from the three basidiomycete fungi Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii to relate their different catalytic capacities to their structural properties. Spectroscopic absorption features and EPR spectra at various pH values of the five enzymes are very similar and typical of the blue oxidases. The analysis of the dependence of kinetic parameters on pH suggested that a histidine residue is involved in the binding of nonphenolic substrates, whereas both a histidine and an acidic residue may be involved in the binding of phenolic compounds. His and an Asp residue are indeed found at the bottom of a cavity which may be regarded as a suitable substrate channel for approaching to type 1 copper in the 3D homology models of the two laccases from Pleuorotus ostreatus (POXC and POXA1b) whose sequences are known.     

Effect of glycosylation on the biochemical properties of <Emphasis Type="Italic">β</Emphasis>-xylosidases from <Emphasis Type="Italic">Aspergillus versicolor</Emphasis>

Aspergillus versicolor grown on xylan or xylose produces two β-xylosidases with differences in biochemical properties and degree of glycosylation. We investigated the alterations in the biochemical properties of these β-xylosidases after deglycosylation with Endo-H or PNGase F. After deglycosylation, both enzymes migrated faster in PAGE or SDS-PAGE exhibiting the same R_f. Temperature optimum of xylan-induced and xylose-induced β-xylosidases was 45°C and 40°C, respectively, and 35°C after deglycosylation. The xylan-induced enzyme was more active at acidic pH. After deglycosylation, both enzymes had the same pH optimum of 6.0. Thermal resistance at 55°C showed half-life of 15 min and 9 min for xylose- and xylan-induced enzymes, respectively. After deglycosylation, both enzymes exhibited half-lives of 7.5 min. Native enzymes exhibited different responses to ions, while deglycosylated enzymes exhibited identical responses. Limited proteolysis yielded similar polypeptide profiles for the deglycosylated enzymes, suggesting a common polypeptide core with differential glycosylation apparently responsible for their biochemical and biophysical differences.     

Genetics and biochemistry of phenol degradation byPseudomonas sp. CF600

Pseudomonas sp. strain CF600 is an efficient degrader of phenol and methylsubstituted phenols. These compounds are degraded by the set of enzymes encoded by the plasmid locateddmpoperon. The sequences of all the fifteen structural genes required to encode the nine enzymes of the catabolic pathway have been determined and the corresponding proteins have been purified. In this review the interplay between the genetic analysis and biochemical characterisation of the catabolic pathway is emphasised. The first step in the pathway, the conversion of phenol to catechol, is catalysed by a novel multicomponent phenol hydroxylase. Here we summarise similarities of this enzyme with other multicomponent oxygenases, particularly methane monooxygenase (EC 1.14.13.25). The other enzymes encoded by the operon are those of the well-knownmeta-cleavage pathway for catechol, and include the recently discoveredmeta-pathway enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10). The known properties of thesemeta-pathway enzymes, and isofunctional enzymes from other aromatic degraders, are summarised. Analysis of the sequences of the pathway proteins, many of which are unique to themeta-pathway, suggests new approaches to the study of these generally little-characterised enzymes. Furthermore, biochemical studies of some of these enzymes suggest that physical associations betweenmeta-pathway enzymes play an important role. In addition to the pathway enzymes, the specific regulator of phenol catabolism, DmpR, and its relationship to the XylR regulator of toluene and xylene catabolism is discussed.     

Construction and characterization of novel chimeric β-glucosidases with Cellvibrio gilvus (CG) and Thermotoga maritima (TM) by overlapping PCR

In order to create novel β-glucosidase constructs, 8 kinds of chimeric β-glucosidases were constructed using overlapping polymerase chain reaction (PCR) based on Cellvibrio gilvus (CG) and Thermotoga maritima (TM) genes. Two kinds of novel chimeric β-glucosidases (No. 6 and No. 8 type) were selected and their properties characterized. SDS-PAGE analysis showed that both constructs had a molecular mass of 80 kDa. The optimum pH of No. 6 chimeric β-glucosidase was found to be 3.0 and 5.0, showing varying maximum activity according to the buffer used. No. 8 chimeric enzyme was found to be optimally active at a pH of 4.5 and the optimum temperature of No.6 and No.8 chimeric β-glucosidases was reported to be 60°C, respectively. The Km values of both novel chimeric enzymes were calculated to be 0.012 mM and 0.0082 mM, respectively and the characteristics of the novel chimeric enzymes were to lie between those of the parental enzymes.     

Purification and characterization of Mycobacterium tuberculosis KatG, KatG(S315T), and Mycobacterium bovis KatG(R463L)

Isoniazid, a first-line antibiotic used for the treatment of tuberculosis, is a prodrug that requires activation by the Mycobacterium tuberculosis enzyme KatG. The KatG(S315T) mutation causes isoniazid resistance while the KatG(R463L) variation is thought to be a polymorphism. Much of the work to date focused on isoniazid activation by KatG has utilized recombinant enzyme overexpressed in Escherichia coli. In this work, native KatG and KatG(S315T) were purified from M. tuberculosis, and KatG(R463L) was purified from Mycobacterium bovis. The native molecular weight, enzymatic activity, optical, resonance Raman, and EPR spectra, K(D) for isoniazid binding, and isoniazid oxidation rates were measured and compared for each native enzyme. Further, the properties of the native enzymes were compared and contrasted with those reported for recombinant KatG, KatG(S315T), and KatG(R463L) in order to assess the ability of the recombinant enzymes to act as good models for the native enzymes.     

Assay and properties of some sulphur enzymes in coastal sands

Summary Arylsulphohydrolase; cysteine desulphohydrase and thiosulphate cyanide sulphurtransferase were assayed in coastal sands and their properties determined. Low enzyme activity was detected in sands lacking vegetation, but much higher activities were detected in the rhizospheres of climax vegetation;Hippopha? rhamnoides andAmmophila sp. Properties of the enzymes were generally similar to those quoted for the enzymes in soils.     

Comparative biochemistry of bacterial N-acyl- -amino acid amidohydrolase

N-acyl- -amino acid amidohydrolases can be classified into three types based on substrate specificity. -aminoacylase has been reported to occur in a very few bacteria such as Pseudomonas, Streptomyces, and Alcaligenes. N-acyl- -aspartate amidohydrolase ( -AAase) has been reported in only Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) while N-acyl- -glutamate amidohydrolase ( -AGase) has been isolated in two stains of Pseudomonas sp. 5f-1 and Alcaligenes A-6. The physiological roles of these enzymes in these microbes are not clear. They are individually characteristic in their substrate specificities, inducer profiles, inhibitors, isoelectric points, metal dependency, and some physicochemical properties. The primary structures of all the three types of N-acyl- -amino acid amidohydrolases from Alcaligenes A-6 were determined from their nucleotide sequences. Comparison of their primary structures revealed high homology (46–56%) between the different enzymes. The three enzymes showed 26–27% sequence homology with -aminoacylases from Bacillus stearothermophilus, porcine, and human. Chemical modification and site-directed mutagenesis identified the histidyl residues essential for catalysis. The Alcaligenes N-acyl- -amino acid amidohydrolases share significant sequence similarities with some members of the urease-related amidohydrolase superfamily proposed by Holm and Sander [L. Holm, C. Sander, Proteins: Structure, Function and Genetics 28 (1997) 72].     

Denitrification and its control

Denitrification in bacteria comprises a series of four reduction reactions; for nitrate, nitrite, nitric oxide and nitrous oxide. Nitrogen gas is the final product. The nature of the enzymes catalysing these reactions is described along with the the properties of the underlying electron transport systems. The factors influencing the expression of the reductases for the four reactions are reviewed along with the effect of oxygen on the activities of the enzymes of denitrification. The main emphasis is on observations made withParacoccus denitrificans andPseudomonas stutzeri.     

Comparative studies of lactic acid dehydrogenases in lactic acid bacteria

The stability, pH-dependence and kinetic properties of the Mn²⁺ and FDP-activated NAD-dependent lactic acid dehydrogenases from Lactobacillus casei ssp. casei (ATCC 393) and L. curvatus (DSM) 20010) were studied after the enzymes were purified to homogeneity by affinity chromatography. Both enzymes are virtually unidirectional, catalysing efficiently only the reduction of pyruvate. They are similar with respect to the effector requirement and pH-optimum. They differ, however, in their electrophoretic mobility, heat stability, pH-dependence of the Mn²⁺ requirement and several kinetic properties. It is suggested that most of these differences are caused by differences of the negative charges in the vicinity of the FDP-binding site or the site responsible for the interaction of the subunits of the enzymatically active oligomeres.Abbreviations l-LDH l-Lactic acid dehydrogenase - FDP Fructose-1,6-bisphosphate - DTE Dithioerythrol AddendumIn the case of the L. casei-LDH the shape of the NADH saturation curve is not changed by omitting the effectors FDP and Mn 2⁺. The K _M under these conditions is 3 fold higher (10.10 ^–5 M).     

PHA synthase engineering toward superbiocatalysts for custom-made biopolymers

Poly-3-hydroxyalkanoates [P(3HA)s] are biologically produced polyesters that have attracted much attention as biodegradable polymers that can be produced from biorenewable resources. These polymers have many attractive properties for use as bulk commodity plastics, fishing lines, and medical uses that are dependent on the repeating unit structures. Despite the readily apparent benefits of using P(3HA)s as replacements for petrochemical-derived plastics, the use and distribution of P(3HA)s have been limited by their cost of production. This problem is currently being addressed by the engineering of enzymes involved in the production of P(3HA)s. Polyhydroxyalkanoate (PHA) synthase (PhaC) enzymes, which catalyze the polymerization of 3-hydroxyacyl-CoA monomers to P(3HA)s, were subjected to various forms of protein engineering to improve the enzyme activity or substrate specificity. This review covers the recent history of PHA synthase engineering and also summarizes studies that have utilized engineered PHA synthases.     

Purification and properties of p-hydroxybenzoate hydroxylases from Rhodococcus strains

Gram-positive bacteria of the genus Rhodococcus catabolize p-hydroxybenzoate (PHB) through the initial formation of 3,4-dihydroxybenzoate. High levels of p-hydroxybenzoate hydroxylase (PHBH) activity are induced in six different Rhodococcus species when these strains are grown on PHB as sole carbon source. The PHBH enzymes were purified to apparent homogeneity and appeared to be homodimers of about 95 kD with each subunit containing a relatively weakly bound FAD. In contrast to their counterparts from gram-negative microorganisms, the Rhodococcus PHBH enzymes prefer NADH to NADPH as external electron donor. All purified enzymes were inhibited by Cl^– and for five of six enzymes more pronounced substrate inhibition was observed in the presence of chloride ions.     

A homologous expression system for obtaining engineered cytochrome ba3 from Thermus thermophilus HB8

Cytochrome ba3 is an integral membrane protein that serves as a terminal oxidase of the respiratory chain in some prokaryotes. We have cloned the complete cba operon of Thermus thermophilus HB8 in an Escherichia coli/T. thermophilus shuttle vector. The ba3-encoding operon, cba, was eliminated from the chromosome of T. thermophilus strain MT111 using the pyrE system of Yamagishi and co-workers. Expression of functional cytochrome ba3 occurred in cells grown at reduced dioxygen levels. A hepta-histidine tag was placed at the N-terminus of subunit I, and a purification method for this form of the enzyme was developed. Growth conditions were investigated for moderate sized cultures (2L) with typical yields of approximately 2 mg of highly pure enzyme per liter of culture medium. The physical properties and enzymatic activities of these recombinant enzymes were compared with those of native enzyme. Recombinant enzyme lacking the histidine tag is spectrally identical to wild-type enzyme. Histidine-tagged cytochrome ba3 shows minor differences from wild-type, and it appears be somewhat less active as a cytochrome c552 oxidase. Exemplary mutants were also produced and compared to native protein. Tyrosine I-237, previously found to be covalently bonded to I-His-233, was changed to phenylalanine (I-Y237F) and to histidine (I-Y237H) in the hepta-histidine tagged cytochrome ba3. The Y to F mutant is devoid of enzyme activity whereas the Y to H mutant possesses approximately 5% wild-type oxidase activity; their properties are compared with those of wild-type enzyme. The above versions of the histidine-tagged enzyme have been crystallized, and our analysis of a 2.3 angstrom resolution electron-density map will be discussed elsewhere.     

Involvement of the ntrA gene product in the anaerobic metabolism of Escherichia coli

Summary The ntrA gene product, required for expression of genes involved in nitrogen fixation (nif) and regulation (ntr), was shown to be necessary for the expression of the two enzymes of the anaerobically inducible formate hydrogenlyase (FHL) pathway, formate dehydrogenase (FDH_H) and hydrogenase isoenzyme 3. Consistent with this finding, the gene encoding the selenopolypeptide (fdhF) of FDH_H was shown to have a nif consensus promoter. The levels of six other anaerobically inducible enzymes were examined and found to be ntrA independent. Significantly, these latter six enzymes are dependent upon the fnr gene product for their expression while FDH_H and hydrogenase 3 are fnr independent. These findings indicate that there are at least two classes of anaerobically regulated promoters: one class which is ntrA dependent and fnr independent and a second class which is fnr dependent and ntrA independent.     

A cluster of esterase genes on chromosome 3R ofDrosophila melanogaster includes homologues of esterase genes conferring insecticide resistance inLucilia cuprina

We identify an esterase isozyme inDrosophila melanogaster, EST 23, which shares biochemical, physiological, and genetic properties with esterase E3, which is involved in resistance to organophosphate insecticides inLucilia cuprina. Like E3, theD. melanogaster EST 23 is a membrane-bound -esterase which migrates slowly toward the anode at pH 6.8. Both enzymes have similar preferences for substrates with shorter acid side chain lengths. Furthermore, on the basis of their high sensitivity to inhibition by paraoxon and their insensitivity to inhibition by eserine sulfate, both enzymes were classified as subclass I carboxylesterases. The activity of each enzyme peaks early in development and, again, in the adult stage. Both enzymes are found in the male reproductive system and larval and adult digestive tissues, the latter being consistent with a role for these enzymes in organophosphate resistance. Fine structure deficiency mapping localizedEst 23 to cytological region 84D3 to E1-2 on the right arm of chromosome 3. Moreover, we show that the genes encoding three other esterase phenotypes also map to the same region; these phenotypes involve allozymic differences in EST 9 (formerly EST C), ali-esterase activity, defined by the hydrolysis of methyl butyrate, and malathion carboxylesterase activity, defined by hydrolysis of the organophosphate malathion. This cluster corresponds closely to that encompassing E3 and malathion carboxylesterase on chromosome 4 inL. cuprina, the homologue of chromosome 3R inD. melanogaster.     

Potential of genes and gene products fromTrichoderma sp. andGliocladium sp. for the development of biological pesticides

Fungal cell wall degrading enzymes produced by the biocontrol fungiTrichoderma harzianum andGliocladium virens are strong inhibitors of spore germination and hyphal elongation of a number of phytopathogenic fungi. The purified enzymes include chitinolytic enzymes with different modes of action or different substrate specificity and glucanolytic enzymes with exo-activity. A variety of synergistic interactions were found when different enzymes were combined or associated with biotic or abiotic antifungal agents. The levels of inhibition obtained by using enzyme combinations were, in some cases, comparable with commercial fungicides. Moreover, the antifungal interaction between enzymes and common fungicides allowed the reduction of the chemical doses up to 200-fold. Chitinolytic and glucanolytic enzymes fromT. harzianum were able to improve substantially the antifungal ability of a biocontrol strain ofEnterobacter cloacae. DNA fragments containing genes encoding for different chitinolytic enzymes were isolated from a cDNA library ofT. harzianum and cloned for mechanistic studies and biocontrol purposes. Our results provide additional information on the role of lytic enzymes in processes of biocontrol and strongly suggest the use of lytic enzymes and their genes for biological control of plant diseases.     

Substrate specificities of a bacterial sialidase and rat liver ganglioside GM3 sialyltransferase

Sialidases cleave off sialic acid residues from the oligosaccharide chain of gangliosides in their catabolic pathway while sialyltransferases transfer sialic acid to the growing oligosaccharide moiety in ganglioside biosynthesis. Ganglioside G_M3 is a common substrate for both types of enzymes, for sialidase acting on ganglioside G_M3 as well as for ganglioside G_D3 synthase. Therefore, it is possible that both enzymes recognize similar structural features of the sialic acid moiety of their common substrate, ganglioside G_M3. Based on this idea we used a variety of G_M3 derivatives as glycolipid substrates for a bacterial sialidase (Clostridium perfringens) and for G_D3 synthase (of rat liver Golgi vesicles). This study revealed that those G_M3 derivatives that were poorly degraded by sialidase also were hardly recognized by sialyltransferase (G_D3 synthase). This may indicate similarities in the substrate binding sites of these enzymes.     

Genetic control of alcohol dehydrogenase levels in Drosophila

Among the progeny of Drosophila flies heterozygous for two noncomplementing Adh-negative alleles, two individuals were found that had recovered appreciable alcohol dehydrogenase activity, thereby surviving the ethanol medium used as a screen. The most likely explanation is that these Adh-positive flies are the product of intracistronic recombination within the Adh locus. Judging by the distribution of outside markers, one of the crossovers would have been a conventional reciprocal exchange while the other appears to have been an instance of nonreciprocal recombination. The enzymes produced in strains derived from the original survivors can be easily distinguished from wild-type enzymes ADH-S and ADH-F on the basis of their sensitivity to denaturing agents. None of various physical and catalytic properties tested revealed differences between the enzymes of the survivor strains except that in one of them the level of activity is 55–65% of the other. Quantitative immunological determinations of ADH gave estimates of enzyme protein which are proportional to the measured activity levels. These results are interpreted to indicate that different amounts of ADH protein are being accumulated in the two strains.This work was supported in part by NSF Grant PCM 76-19563.