Interactions Outside the Proteinase-binding Loop Contribute Significantly to the Inhibition of Activated Coagulation Factor XII by Its Canonical Inhibitor from Corn

Activated factor XII (FXIIa) is selectively inhibited by corn Hageman factor inhibitor (CHFI) among other plasma proteases. CHFI is considered a canonical serine protease inhibitor that interacts with FXIIa through its protease-binding loop. Here we examined whether the protease-binding loop alone is sufficient for the selective inhibition of serine proteases or whether other regions of a canonical inhibitor are involved. Six CHFI mutants lacking different N- and C-terminal portions were generated. CHFI-234, which lacks the first and fifth disulfide bonds and 11 and 19 amino acid residues at the N and C termini, respectively, exhibited no significant changes in FXIIa inhibition (K_i = 3.2 ± 0.4 nm). CHFI-123, which lacks 34 amino acid residues at the C terminus and the fourth and fifth disulfide bridges, inhibited FXIIa with a K_i of 116 ± 16 nm. To exclude interactions outside the FXIIa active site, a synthetic cyclic peptide was tested. The peptide contained residues 20–45 (Protein Data Bank code 1BEA), and a C29D substitution was included to avoid unwanted disulfide bond formation between unpaired cysteines. Surprisingly, the isolated protease-binding loop failed to inhibit FXIIa but retained partial inhibition of trypsin (K_i = 11.7 ± 1.2 μm) and activated factor XI (K_i = 94 ± 11 μm). Full-length CHFI inhibited trypsin with a K_i of 1.3 ± 0.2 nm and activated factor XI with a K_i of 5.4 ± 0.2 μm. Our results suggest that the protease-binding loop is not sufficient for the interaction between FXIIa and CHFI; other regions of the inhibitor also contribute to specific inhibition.     

Crystal Structure of an Exo-1,5-α-l-arabinofuranosidase from Streptomyces avermitilis Provides Insights into the Mechanism of Substrate Discrimination between Exo- and Endo-type Enzymes in Glycoside Hydrolase Family 43

Exo-1,5-α-l-arabinofuranosidases belonging to glycoside hydrolase family 43 have strict substrate specificity. These enzymes hydrolyze only the α-1,5-linkages of linear arabinan and arabino-oligosaccharides in an exo-acting manner. The enzyme from Streptomyces avermitilis contains a core catalytic domain belonging to glycoside hydrolase family 43 and a C-terminal arabinan binding module belonging to carbohydrate binding module family 42. We determined the crystal structure of intact exo-1,5-α-l-arabinofuranosidase. The catalytic module is composed of a 5-bladed β-propeller topologically identical to the other family 43 enzymes. The arabinan binding module had three similar subdomains assembled against one another around a pseudo-3-fold axis, forming a β-trefoil-fold. A sugar complex structure with α-1,5-l-arabinofuranotriose revealed three subsites in the catalytic domain, and a sugar complex structure with α-l-arabinofuranosyl azide revealed three arabinose-binding sites in the carbohydrate binding module. A mutagenesis study revealed that substrate specificity was regulated by residues Asn-159, Tyr-192, and Leu-289 located at the aglycon side of the substrate-binding pocket. The exo-acting manner of the enzyme was attributed to the strict pocket structure of subsite −1, formed by the flexible loop region Tyr-281–Arg-294 and the side chain of Tyr-40, which occupied the positions corresponding to the catalytic glycon cleft of GH43 endo-acting enzymes.     

Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K_m for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm.     

Novel Enzyme Family Found in Filamentous Fungi Catalyzing trans-4-Hydroxylation of l-Pipecolic Acid

Hydroxypipecolic acids are bioactive compounds widely distributed in nature and are valuable building blocks for the organic synthesis of pharmaceuticals. We have found a novel hydroxylating enzyme with activity toward l-pipecolic acid (l-Pip) in a filamentous fungus, Fusarium oxysporum c8D. The enzyme l-Pip trans-4-hydroxylase (Pip4H) of F. oxysporum (FoPip4H) belongs to the Fe(II)/α-ketoglutarate-dependent dioxygenase superfamily, catalyzes the regio- and stereoselective hydroxylation of l-Pip, and produces optically pure trans-4-hydroxy-l-pipecolic acid (trans-4-l-HyPip). Amino acid sequence analysis revealed several fungal enzymes homologous with FoPip4H, and five of these also had l-Pip trans-4-hydroxylation activity. In particular, the homologous Pip4H enzyme derived from Aspergillus nidulans FGSC A4 (AnPip4H) had a broader substrate specificity spectrum than other homologues and reacted with the l and d forms of various cyclic and aliphatic amino acids. Using FoPip4H as a biocatalyst, a system for the preparative-scale production of chiral trans-4-l-HyPip was successfully developed. Thus, we report a fungal family of l-Pip hydroxylases and the enzymatic preparation of trans-4-l-HyPip, a bioactive compound and a constituent of secondary metabolites with useful physiological activities.     

Crystal Structure and Mutational Analysis of Aminoacylhistidine Dipeptidase from Vibrio alginolyticus Reveal a New Architecture of M20 Metallopeptidases

Aminoacylhistidine dipeptidases (PepD, EC 3.4.13.3) belong to the family of M20 metallopeptidases from the metallopeptidase H clan that catalyze a broad range of dipeptide and tripeptide substrates, including l-carnosine and l-homocarnosine. Homocarnosine has been suggested as a precursor for the neurotransmitter γ-aminobutyric acid (GABA) and may mediate the antiseizure effects of GABAergic therapies. Here, we report the crystal structure of PepD from Vibrio alginolyticus and the results of mutational analysis of substrate-binding residues in the C-terminal as well as substrate specificity of the PepD catalytic domain-alone truncated protein PepD^CAT. The structure of PepD was found to exist as a homodimer, in which each monomer comprises a catalytic domain containing two zinc ions at the active site center for its hydrolytic function and a lid domain utilizing hydrogen bonds between helices to form the dimer interface. Although the PepD is structurally similar to PepV, which exists as a monomer, putative substrate-binding residues reside in different topological regions of the polypeptide chain. In addition, the lid domain of the PepD contains an “extra” domain not observed in related M20 family metallopeptidases with a dimeric structure. Mutational assays confirmed both the putative di-zinc allocations and the architecture of substrate recognition. In addition, the catalytic domain-alone truncated PepD^CAT exhibited substrate specificity to l-homocarnosine compared with that of the wild-type PepD, indicating a potential value in applications of PepD^CAT for GABAergic therapies or neuroprotection.     

Structural Basis for Competitive Inhibition of 3,4-Dihydroxy-2-butanone-4-phosphate Synthase from Vibrio cholerae

The riboflavin biosynthesis pathway has been shown to be essential in many pathogens and is absent in humans. Therefore, enzymes involved in riboflavin synthesis are considered as potential antibacterial drug targets. The enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes one of the two committed steps in the riboflavin pathway and converts d-ribulose 5-phosphate (Ru5P) to l-3,4-dihydroxy-2-butanone 4-phosphate and formate. Moreover, DHBPS is shown to be indispensable for Mycobacterium, Salmonella, and Helicobacter species. Despite the essentiality of this enzyme in bacteria, no inhibitor has been identified hitherto. Here, we describe kinetic and crystal structure characterization of DHBPS from Vibrio cholerae (vDHBPS) with a competitive inhibitor 4-phospho-d-erythronohydroxamic acid (4PEH) at 1.86-Å resolution. In addition, we also report the structural characterization of vDHBPS in its apo form and in complex with its substrate and substrate plus metal ions at 1.96-, 1.59-, and 2.04-Å resolution, respectively. Comparison of these crystal structures suggests that 4PEH inhibits the catalytic activity of DHBPS as it is unable to form a proposed intermediate that is crucial for DHBPS activity. Furthermore, vDHBPS structures complexed with substrate and metal ions reveal that, unlike Candida albicans, binding of substrate to vDHBPS induces a conformational change from an open to closed conformation. Interestingly, the position of second metal ion, which is different from that of Methanococcus jannaschii, strongly supports an active role in the catalytic mechanism. Thus, the kinetic and structural characterization of vDHBPS reveals the molecular mechanism of inhibition shown by 4PEH and that it can be explored further for designing novel antibiotics.     

Crystal Structure and Characterization of the Glycoside Hydrolase Family 62 α-l-Arabinofuranosidase from Streptomyces coelicolor

α-l-Arabinofuranosidase, which belongs to the glycoside hydrolase family 62 (GH62), hydrolyzes arabinoxylan but not arabinan or arabinogalactan. The crystal structures of several α-l-arabinofuranosidases have been determined, although the structures, catalytic mechanisms, and substrate specificities of GH62 enzymes remain unclear. To evaluate the substrate specificity of a GH62 enzyme, we determined the crystal structure of α-l-arabinofuranosidase, which comprises a carbohydrate-binding module family 13 domain at its N terminus and a catalytic domain at its C terminus, from Streptomyces coelicolor. The catalytic domain was a five-bladed β-propeller consisting of five radially oriented anti-parallel β-sheets. Sugar complex structures with l-arabinose, xylotriose, and xylohexaose revealed five subsites in the catalytic cleft and an l-arabinose-binding pocket at the bottom of the cleft. The entire structure of this GH62 family enzyme was very similar to that of glycoside hydrolase 43 family enzymes, and the catalytically important acidic residues found in family 43 enzymes were conserved in GH62. Mutagenesis studies revealed that Asp²⁰² and Glu³⁶¹ were catalytic residues, and Trp²⁷⁰, Tyr⁴⁶¹, and Asn⁴⁶² were involved in the substrate-binding site for discriminating the substrate structures. In particular, hydrogen bonding between Asn⁴⁶² and xylose at the nonreducing end subsite +2 was important for the higher activity of substituted arabinofuranosyl residues than that for terminal arabinofuranoses.     

Site-directed Mutagenesis Switching a Dimethylallyl Tryptophan Synthase to a Specific Tyrosine C3-Prenylating Enzyme

The tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl tryptophan synthase) from Aspergillus fumigatus catalyze C⁴- and C⁷-prenylation of the indole ring, respectively. 7-DMATS was found to accept l-tyrosine as substrate as well and converted it to an O-prenylated derivative. An acceptance of l-tyrosine by FgaPT2 was also observed in this study. Interestingly, isolation and structure elucidation revealed the identification of a C³-prenylated l-tyrosine as enzyme product. Molecular modeling and site-directed mutagenesis led to creation of a mutant FgaPT2_K174F, which showed much higher specificity toward l-tyrosine than l-tryptophan. Its catalytic efficiency toward l-tyrosine was found to be 4.9-fold in comparison with that of non-mutated FgaPT2, whereas the activity toward l-tryptophan was less than 0.4% of that of the wild-type. To the best of our knowledge, this is the first report on an enzymatic C-prenylation of l-tyrosine as free amino acid and altering the substrate preference of a prenyltransferase by mutagenesis.     

Microneme Protein 5 Regulates the Activity of Toxoplasma Subtilisin 1 by Mimicking a Subtilisin Prodomain

Toxoplasma gondii is the model parasite of the phylum Apicomplexa, which contains obligate intracellular parasites of medical and veterinary importance. Apicomplexans invade host cells by a multistep process involving the secretion of adhesive microneme protein (MIC) complexes. The subtilisin protease TgSUB1 trims several MICs on the parasite surface to activate gliding motility and host invasion. Although a previous study showed that expression of the secretory protein TgMIC5 suppresses TgSUB1 activity, the mechanism was unknown. Here, we solve the three-dimensional structure of TgMIC5 by nuclear magnetic resonance (NMR), revealing that it mimics a subtilisin prodomain including a flexible C-terminal peptide that may insert into the subtilisin active site. We show that TgMIC5 is an almost 50-fold more potent inhibitor of TgSUB1 activity than the small molecule inhibitor N-[N-(N-acetyl-l-leucyl)-l-leucyl]-l-norleucine (ALLN). Moreover, we demonstrate that TgMIC5 is retained on the parasite plasma membrane via its physical interaction with the membrane-anchored TgSUB1.     

A Novel Type of Peptidoglycan-binding Domain Highly Specific for Amidated d-Asp Cross-bridge,Identified in Lactobacillus casei Bacteriophage Endolysins

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-l-alanine amidase, whereas Lc-Lys-2 is a γ-d-glutamyl-l-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with d-Ala⁴→d-Asx-l-Lys³ in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting d-Ala⁴→l-Ala-(l-Ala/l-Ser)-l-Lys³; moreover, they do not lyse the L. lactis mutant containing only the nonamidated d-Asp cross-bridge, i.e. d-Ala⁴→d-Asp-l-Lys³. In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 l-Lys³-d-Asn-l-Lys³ bridges replacing the wild-type 4→3 d-Ala⁴-d-Asn-l-Lys³ bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly d-Asn but not PG with only the nonamidated d-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the d-Asn interpeptide bridge of PG.     

Recharacterization of the mammalian cytosolic type 2 (R)-β-hydroxybutyrate dehydrogenase as 4-oxo-l-proline reductase (EC 1.1.1.104)

Early studies revealed that chicken embryos incubated with a rare analog of l-proline, 4-oxo-l-proline, showed increased levels of the metabolite 4-hydroxy-l-proline. In 1962, 4-oxo-l-proline reductase, an enzyme responsible for the reduction of 4-oxo-l-proline, was partially purified from rabbit kidneys and characterized biochemically. However, only recently was the molecular identity of this enzyme solved. Here, we report the purification from rat kidneys, identification, and biochemical characterization of 4-oxo-l-proline reductase. Following mass spectrometry analysis of the purified protein preparation, the previously annotated mammalian cytosolic type 2 (R)-β-hydroxybutyrate dehydrogenase (BDH2) emerged as the only candidate for the reductase. We subsequently expressed rat and human BDH2 in Escherichia coli, then purified it, and showed that it catalyzed the reversible reduction of 4-oxo-l-proline to cis-4-hydroxy-l-proline via chromatographic and tandem mass spectrometry analysis. Specificity studies with an array of compounds carried out on both enzymes showed that 4-oxo-l-proline was the best substrate, and the human enzyme acted with 12,500-fold higher catalytic efficiency on 4-oxo-l-proline than on (R)-β-hydroxybutyrate. In addition, human embryonic kidney 293T (HEK293T) cells efficiently metabolized 4-oxo-l-proline to cis-4-hydroxy-l-proline, whereas HEK293T BDH2 KO cells were incapable of producing cis-4-hydroxy-l-proline. Both WT and KO HEK293T cells also produced trans-4-hydroxy-l-proline in the presence of 4-oxo-l-proline, suggesting that the latter compound might interfere with the trans-4-hydroxy-l-proline breakdown in human cells. We conclude that BDH2 is a mammalian 4-oxo-l-proline reductase that converts 4-oxo-l-proline to cis-4-hydroxy-l-proline and not to trans-4-hydroxy-l-proline, as originally thought. We also hypothesize that this enzyme may be a potential source of cis-4-hydroxy-l-proline in mammalian tissues.     

Engineering Filamentous Fungi for Conversion of d-Galacturonic Acid to l-Galactonic Acid

d-Galacturonic acid, the main monomer of pectin, is an attractive substrate for bioconversions, since pectin-rich biomass is abundantly available and pectin is easily hydrolyzed. l-Galactonic acid is an intermediate in the eukaryotic pathway for d-galacturonic acid catabolism, but extracellular accumulation of l-galactonic acid has not been reported. By deleting the gene encoding l-galactonic acid dehydratase (lgd1 or gaaB) in two filamentous fungi, strains were obtained that converted d-galacturonic acid to l-galactonic acid. Both Trichoderma reesei Δlgd1 and Aspergillus niger ΔgaaB strains produced l-galactonate at yields of 0.6 to 0.9 g per g of substrate consumed. Although T. reesei Δlgd1 could produce l-galactonate at pH 5.5, a lower pH was necessary for A. niger ΔgaaB. Provision of a cosubstrate improved the production rate and titer in both strains. Intracellular accumulation of l-galactonate (40 to 70 mg g biomass⁻¹) suggested that export may be limiting. Deletion of the l-galactonate dehydratase from A. niger was found to delay induction of d-galacturonate reductase and overexpression of the reductase improved initial production rates. Deletion of the l-galactonate dehydratase from A. niger also delayed or prevented induction of the putative d-galacturonate transporter An14g04280. In addition, A. niger ΔgaaB produced l-galactonate from polygalacturonate as efficiently as from the monomer.     

The Allosteric Regulatory Mechanism of the Escherichia coli MetNI Methionine ATP Binding Cassette (ABC) Transporter

The MetNI methionine importer of Escherichia coli, an ATP binding cassette (ABC) transporter, uses the energy of ATP binding and hydrolysis to catalyze the high affinity uptake of d- and l-methionine. Early in vivo studies showed that the uptake of external methionine is repressed by the level of the internal methionine pool, a phenomenon termed transinhibition. Our understanding of the MetNI mechanism has thus far been limited to a series of crystal structures in an inward-facing conformation. To understand the molecular mechanism of transinhibition, we studied the kinetics of ATP hydrolysis using detergent-solubilized MetNI. We find that transinhibition is due to noncompetitive inhibition by l-methionine, much like a negative feedback loop. Thermodynamic analyses revealed two allosteric methionine binding sites per transporter. This quantitative analysis of transinhibition, the first to our knowledge for a structurally defined transporter, builds upon the previously proposed structurally based model for regulation. This mechanism of regulation at the transporter activity level could be applicable to not only ABC transporters but other types of membrane transporters as well.     

Substrate and Reaction Specificity of Mycobacterium tuberculosis Cytochrome P450 CYP121: INSIGHTS FROM BIOCHEMICAL STUDIES AND CRYSTAL STRUCTURES*

Cytochrome P450 CYP121 is essential for the viability of Mycobacterium tuberculosis. Studies in vitro show that it can use the cyclodipeptide cyclo(l-Tyr-l-Tyr) (cYY) as a substrate. We report an investigation of the substrate and reaction specificities of CYP121 involving analysis of the interaction between CYP121 and 14 cYY analogues with various modifications of the side chains or the diketopiperazine (DKP) ring. Spectral titration experiments show that CYP121 significantly bound only cyclodipeptides with a conserved DKP ring carrying two aryl side chains in l-configuration. CYP121 did not efficiently or selectively transform any of the cYY analogues tested, indicating a high specificity for cYY. The molecular determinants of this specificity were inferred from both crystal structures of CYP121-analog complexes solved at high resolution and solution NMR spectroscopy of the analogues. Bound cYY or its analogues all displayed a similar set of contacts with CYP121 residues Asn⁸⁵, Phe¹⁶⁸, and Trp¹⁸². The propensity of the cYY tyrosyl to point toward Arg³⁸⁶ was dependent on the presence of the DKP ring that limits the conformational freedom of the ligand. The correct positioning of the hydroxyl of this tyrosyl was essential for conversion of cYY. Thus, the specificity of CYP121 results from both a restricted binding specificity and a fine-tuned P450 substrate relationship. These results document the catalytic mechanism of CYP121 and improve our understanding of its function in vivo. This work contributes to progress toward the design of inhibitors of this essential protein of M. tuberculosis that could be used for antituberculosis therapy.     

A Novel Bacterial Enzyme with d-Glucuronyl C5-epimerase Activity

Glycosaminoglycans are biologically active polysaccharides that are found ubiquitously in the animal kingdom. The biosynthesis of these complex polysaccharides involves complicated reactions that turn the simple glycosaminoglycan backbone into highly heterogeneous structures. One of the modification reactions is the epimerization of d-glucuronic acid to its C5-epimer l-iduronic acid, which is essential for the function of heparan sulfate. Although l-iduronic acid residues have been shown to exist in polysaccharides of some prokaryotes, there has been no experimental evidence for the existence of a prokaryotic d-glucuronyl C5-epimerase. This work for the first time reports on the identification of a bacterial enzyme with d-glucuronyl C5-epimerase activity. A gene of the marine bacterium Bermanella marisrubri sp. RED65 encodes a protein (RED65_08024) of 448 amino acids that has an overall 37% homology to the human d-glucuronic acid C5-epimerase. Alignment of this peptide with the human and mouse sequences revealed a 60% similarity at the carboxyl terminus. The recombinant protein expressed in Escherichia coli showed epimerization activity toward substrates generated from heparin and the E. coli K5 capsular polysaccharide, thereby providing the first evidence for bacterial d-glucuronyl C5-epimerase activity. These findings may eventually be used for modification of mammalian glycosaminoglycans.     

Proteases and Peptidases of Castor Bean Endosperm: Enzyme Characterization and Changes during Germination

The endosperm of castor bean seeds (Ricinus communis L.) contains two —SH-dependent aminopeptidases, one hydrolyzing l-leucine-β-naphthylamide optimally at pH 7.0, and the other hydrolyzing l-proline-β-naphthylamide optimally at pH 7.5. After germination the endosperm contains in addition an —SH-dependent hemoglobin protease, a serine-dependent carboxypeptidase, and at least two —SH-dependent enzymes hydrolyzing the model substrate α-N-benzoyl-dl-arginine-β-naphthylamide (BANA). The carboxypeptidase is active on a variety of N-carbobenzoxy dipeptides, especially N-carbobenzoxy-L-phenylalanine-l-alanine and N-carbobenzoxy-l-tyrosine-l-leucine. The pH optima for the protease, carboxypeptidase, and BANAase acivities are 3.5 to 4.0, 5.0 to 5.5, and 6 to 8, respectively.     

Probing the Molecular Determinant for the Catalytic Efficiency of l-Arabinose Isomerase from Bacillus licheniformis

Bacillus licheniformis l-arabinose isomerase (l-AI) is distinguished from other l-AIs by its high degree of substrate specificity for l-arabinose and its high turnover rate. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis to alter individual screened residues, was used to study the molecular determinants for the catalytic efficiency of B. licheniformis l-AI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type B. licheniformis l-AI was identified as an important residue affecting the catalytic efficiency of B. licheniformis l-AI. Further insights into the function of residue Y333 were obtained by replacing it with other aromatic, nonpolar hydrophobic amino acids or polar amino acids. Replacing Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward l-arabinose. In contrast, the activities of mutants containing a hydrophobic amino acid (Ala, Val, or Leu) at position 333 decreased as the size of the hydrophobic side chain of the amino acid decreased. However, mutants containing hydrophilic and charged amino acids, such as Asp, Glu, and Lys, showed almost no activity with l-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in the catalytic efficiency of B. licheniformis l-AI.l-Arabinose isomerase (l-AI) is an enzyme that mediates in vivo isomerization between l-arabinose and l-ribulose as well as in vitro isomerization of d-galactose and d-tagatose (20). l-Ribulose (l-erythro-pentulose) is a rare and expensive ketopentose sugar (1) that can be used as a precursor for the production of other rare sugars of high market value, such as l-ribose. Despite being a common metabolic intermediate in different organisms, l-ribulose is scarce in nature. The market for rare and unnatural sugars has been growing, especially in the sweetener and pharmaceutical industries. For example, several modified nucleosides derived from l-sugars have been shown to act as potent antiviral agents and are also useful in antigen therapy. Derivatives of rare sugars have also been used as agents against hepatitis B virus and human immunodeficiency virus (2, 22).For these reasons, interest in the enzymology of rare sugars has also been increasing. Various forms of l-AI from a variety of organisms have been characterized, and some have shown potential for industrial use. Several highly thermotolerant enzyme forms from Thermotoga maritima (12), Thermotoga neapolitana (10), Bacillus stearothermophilus (18), Thermoanaerobacter mathranii (9), and Lactobacillus plantarum (5) have been characterized previously. All of these reported l-AIs tend to have broad specificity, although a few l-AIs with high degrees of substrate specificity for l-arabinose have also been documented.The enzyme properties of l-AIs have been examined by engineering several forms by error-prone PCR and site-directed mutagenesis. Galactose conversion was reportedly enhanced 20% following site-directed introduction of a double mutation (C450S-N475K) into l-AI (16). Error-prone PCR manipulation of l-AI from Geobacillus stearothermophilus resulted in a shift in temperature specificity from 60 to 65°C and increased isomerization activity (11). All of these previously reported mutational studies have been aimed at improving enzymatic properties for industrial application. However, even though the three-dimensional (3D) structure of Escherichia coli l-AI has been determined previously (15), few new structural studies have been performed to decipher the reaction mechanism of this enzyme. Rhimi et al. (19) have reported an important role for D308, F329, E351, and H446 in catalysis, as indicated by findings from site-directed mutagenesis. Nonetheless, detailed analysis of the important molecular determinants controlling the catalytic activities of the l-AIs is still lacking.Previously, we have reported the cloning and characterization of a novel l-AI from Bacillus licheniformis (17). This enzyme can be distinguished from other l-AIs by its wide pH range, high degree of substrate specificity for l-arabinose, and extremely high turnover rate. In the present paper, we report the identification of an important amino acid residue responsible for the catalytic efficiency of l-AIs, as determined by a systematic screening process composed of sequence alignment and molecular dynamics (MD) simulation, followed by site-directed mutagenesis. Using the crystal structure of E. coli l-AI as a template, we have built a 3D model of B. licheniformis l-AI. Analysis of the 3D model of B. licheniformis l-AI docked with l-arabinose, followed by a systematic screening process, showed that Y333 interacted with the substrate, suggesting that this residue in B. licheniformis l-AI may be essential for catalysis. We further characterized the role of Y333 in B. licheniformis l-AI binding of and catalytic efficiency for l-arabinose.     

Properties of an Aminotransferase of Pea (Pisum sativum L.)

A transaminase (aminotransferase, EC 2.6.1) fraction was partially purified from shoot tips of pea (Pisum sativum L. cv. Alaska) seedlings. With α-ketoglutarate as co-substrate, the enzyme transaminated the following aromatic amino acids: d,l-tryptophan, d,l-tyrosine, and d,l-phenylalanine, as well as the following aliphatic amino acids: d,l-alanine, d,l-methionine, and d,l-leucine. Of other α-keto acids tested, pyruvate and oxalacetate were more active than α-ketoglutarate with d,l-tryptophan. Stoichiometric yields of indolepyruvate and glutamate were obtained with d,l-tryptophan and α-ketoglutarate as co-substrates. The specific activity was three times higher with d-tryptophan than with l-tryptophan.     

Metabolism of Pentose Sugars in the Hyperthermophilic Archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius

We have previously shown that the hyperthermophilic archaeon, Sulfolobus solfataricus, catabolizes d-glucose and d-galactose to pyruvate and glyceraldehyde via a non-phosphorylative version of the Entner-Doudoroff pathway. At each step, one enzyme is active with both C6 epimers, leading to a metabolically promiscuous pathway. On further investigation, the catalytic promiscuity of the first enzyme in this pathway, glucose dehydrogenase, has been shown to extend to the C5 sugars, d-xylose and l-arabinose. In the current paper we establish that this promiscuity for C6 and C5 metabolites is also exhibited by the third enzyme in the pathway, 2-keto-3-deoxygluconate aldolase, but that the second step requires a specific C5-dehydratase, the gluconate dehydratase being active only with C6 metabolites. The products of this pathway for the catabolism of d-xylose and l-arabinose are pyruvate and glycolaldehyde, pyruvate entering the citric acid cycle after oxidative decarboxylation to acetyl-coenzyme A. We have identified and characterized the enzymes, both native and recombinant, that catalyze the conversion of glycolaldehyde to glycolate and then to glyoxylate, which can enter the citric acid cycle via the action of malate synthase. Evidence is also presented that similar enzymes for this pentose sugar pathway are present in Sulfolobus acidocaldarius, and metabolic tracer studies in this archaeon demonstrate its in vivo operation in parallel with a route involving no aldol cleavage of the 2-keto-3-deoxy-pentanoates but direct conversion to the citric acid cycle C5-metabolite, 2-oxoglutarate.     

Isolation and Characterization of Two Momordins,Ribosome-Inactivating Proteins from the Seeds of Bitter Gourd (Momordica charantia)

Cathepsins L and L-like (58 kDa) proteinase from mackerel were purified to electrophoretical homogeneity by Concanavalin A-Sepharose and Econo-Pac S chromatographies. The molecular weights of cathepsins L and L-like proteinase were 30,000 and 58,000, and the optimal pH for the hydrolysis of Z-Phe-Arg-MCA (benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-[4-methyl] coumarylamide) were 5.0 and 5.5, respectively. The stability of both purified proteinases at various pHs was low, when the pH was above 7.0. According to the substrate specificity analysis, these proteinases hydrolyzed Z-Phe-Arg-MCA and Z-Arg-Arg-MCA, but did not hydrolyze Z-Arg-MCA and L-Arg-MCA. The activities of these two proteinases were effectively activated by cysteine and dithiothreitol. Their thiol-dependent proteolytic activity against Z-Phe-Arg-MCA was strongly inhibited by E-64 (trans-epoxysuccinyl-L-leucylamido[4-guanidino]butane), antipain, chymostatin, iodoacetic acid, and leupeptin, but not inhibited by pepstatin or phenylmethane sulfonyl floride. The inactivation rate constants (K_D) of cathepsins L and L-like proteinases at 50°C were 5.1 × 10^?5 and 6.9 × 10^?4 s^?1, respectively. K⁺, Na⁺, Mg⁺, and Sr⁺ did not affect them, while Zn²⁺, Cd²⁺, Co²⁺, Ni²⁺, Cu²⁺, Hg²⁺, Fe²⁺, and Fe³⁺ inhibited the activity of the purified cathepsins L and L-like proteinase.