Cytochromes P450: History, Classes, Catalytic Mechanism, and Industrial Application.

Cytochromes P450, a family of heme-containing monooxygenases that catalyze a diverse range of oxidative reactions, are so-called due to their maximum absorbance at 450nm, ie, "Pigment-450nm," when bound to carbon monoxide. They have appeal both academically and commercially due to their high degree of regio- and stereoselectivity, for example, in the area of active pharmaceutical ingredient synthesis. Despite this potential, they often exhibit poor stability, low turnover numbers and typically require electron transport protein(s) for catalysis. P450 systems exist in a variety of functional domain architectures, organized into 10 classes. P450s are also divided into families, each of which is based solely on amino acid sequence homology. Their catalytic mechanism employs a very complex, multistep catalytic cycle involving a range of transient intermediates. Mutagenesis is a powerful tool for the development of improved biocatalysts and has been used extensively with the archetypal Class VIII P450, BM3, from Bacillus megaterium, but with the increasing scale of genomic sequencing, a huge resource is now available for the discovery of novel P450s.

The structure of the feruloyl esterase module of xylanase 10B from Clostridium thermocellum provides insights into substrate recognition.

BACKGROUND: Degradation of the plant cell wall requires the synergistic action of a consortium of predominantly modular enzymes. In Clostridiae, these biocatalysts are organized into a supramolecular assembly termed a "cellulosome." This multienzyme complex possesses, in addition to its well-described cellulolytic activity, an apparatus specific for xylan degradation. Cinnamic acid esterases hydrolyze the ferulate groups involved in the crosslinking of arabinoxylans to lignin and thus play a key role in the degradation of the plant cell wall in addition to having promising industrial and medical applications. RESULTS: We have cloned and overexpressed the feruloyl esterase module from a 5 domain xylanase, Xyn10B from Clostridium thermocellum. The native structure at 1.6 A resolution has been solved with selenomethionine multiple wavelength anomalous dispersion and refined to a final R(free) of 17.8%. The structure of a hydrolytically inactive mutant, S954A, in complex with the reaction product ferulic acid has been refined at a resolution of 1.4 A with an R(free) of 16.0%. CONCLUSIONS: The C. thermocellum Xyn10B ferulic acid esterase displays the alpha/beta-hydrolase fold and possesses a classical Ser-His-Asp catalytic triad. Ferulate esterases are characterized by their specificity, and the active center reveals the binding site for ferulic acid and related compounds. Ferulate binds in a small surface depression that possesses specificity determinants for both the methoxy and hydroxyl ring substituents of the substrate. There appears to be a lack of specificity for the xylan backbone, which may reflect the intrinsic chemical heterogeneity of the natural substrate.

Three-dimensional structures of the Mn and Mg dTDP complexes of the family GT-2 glycosyltransferase SpsA: a comparison with related NDP-sugar glycosyltransferases.

The vast majority of glycosidic-bond synthesis in nature is performed by glycosyltransferases, which use activated glycosides as the sugar donor. Typically, the activated leaving group is a nucleoside phosphate, lipid phosphate or phosphate. The nucleotide-sugar-dependent glycosyltransferases fall into over 50 sequence-based families, with the largest and most widespread family of inverting transferases named family GT-2. Here, we present the three-dimensional crystal structure of SpsA, the first and currently the only structural representative from family GT-2, in complex with both Mn-dTDP and Mg-dTDP at a resolution of 2 A. These structures reveal how SpsA and related enzymes may display nucleotide plasticity and permit a comparison of the catalytic centre of this enzyme with those from related sequence families whose three-dimensional structures have recently been determined. Family GT-2 enzymes, together with enzymes from families 7, 13 and 43, appear to form a clan of related structures with identical catalytic apparatus and reaction mechanism.

Structure of a family 15 carbohydrate-binding module in complex with xylopentaose. Evidence that xylan binds in an approximate 3-fold helical conformation.

The recycling of photosynthetically fixed carbon by the action of microbial glycoside hydrolases is a key biological process. The consortium of degradative enzymes involved in this process frequently display catalytic modules appended to one or more noncatalytic carbohydrate-binding modules (CBMs). CBMs play a central role in the optimization of the catalytic activity of plant cell wall hydrolases through their binding to specific plant structural polysaccharides. Despite their pivotal role in the biodegradation of plant biomass, the mechanism by which these proteins recognize their target ligands is unclear. This report describes the structure of a xylan-binding CBM (CBM15) in complex with its ligand. This module, derived from Pseudomonas cellulosa xylanase Xyn10C, binds to both soluble xylan and xylooligosaccharides. The three-dimensional crystal structure of CBM15 bound to xylopentaose has been solved by x-ray crystallography to a resolution of 1.6 A. The protein displays a similar beta-jelly roll fold to that observed in many other families of binding-modules. A groove, 20-25 A in length, on the concave surface of one of the beta-sheets presents two tryptophan residues, the faces of which are orientated at approximately 240 degrees to one another. These form-stacking interactions with the n and n+2 sugars of xylopentaose complementing the approximate 3-fold helical structure of this ligand in the binding cleft of CBM15. In four of the five observed binding subsites, the 2' and 3' hydroxyls of the bound ligand are solvent-exposed, providing an explanation for the capacity of this xylan-binding CBM to accommodate the highly decorated xylans found in the plant cell wall.

Clostridium thermocellum Xyn10B carbohydrate-binding module 22-2: the role of conserved amino acids in ligand binding.

The majority of plant cell wall hydrolases are modular enzymes which, in addition to a catalytic module, possess one or more carbohydrate-binding modules (CBMs). These carbohydrate-active enzymes and their constituent modules have been classified into a number of families based upon amino acid sequence similarity. The Clostridium thermocellum xylanase, Xyn10B, contains two CBMs that belong to family 22 (CBM22). The crystal structure of the C-terminal CBM22 (CBM22-2) was determined in a previous study [Charnock, S. J., et al. (2000) Biochemistry 39, 5013--5021] and revealed a surface cleft which presents several conserved residues that are implicated in ligand binding. These amino acids have been substituted and the structure and biochemical properties of the mutants analyzed. The data show that R25A, W53A, Y103A, Y136A, and E138A exhibit greatly reduced affinity for xylotetraose relative to that of the wild-type protein. Conversely, mutations Y103F and Y136F have little effect on ligand binding. Using thermodynamic, X-ray, and NMR measurements on the mutants, we show that the cleft of CBM22-2 does indeed form the ligand-binding site. Trp 53 and Tyr 103 most likely participate in hydrophobic stacking interactions with the ligand, while Glu 138 makes one or more important hydrogen bonds with the tetrasaccharide. Although Arg 25 and Tyr 136 are likely to form hydrogen bonds with the ligand, they are also shown to play a critical role in maintaining the structural integrity of the binding cleft.

Cloning, purification and characterization of the 6-phospho-3-hexulose isomerase YckF from Bacillus subtilis.

The enzyme 6-phospho-3-hexulose isomerase (YckF) from Bacillus subtilis has been prepared and crystallized in a form suitable for X-ray crystallographic analysis. Crystals were grown by the hanging-drop method at 291 K using polyethylene glycol 2000 monomethylether as precipitant. They diffract beyond 1.7 A using an in-house Cu Kalpha source and belong to either space group P6(5)22 or P6(1)22, with unit-cell parameters a = b = 72.4, c = 241.2 A, and have two molecules of YckF in the asymmetric unit.

Characterization of a novel pectate lyase, Pel10A, from Pseudomonas cellulosa.

Biological recycling of plant material is essential for biosphere maintenance. This perpetual task involves a complex array of enzymes, including extracellular polysaccharide hydrolases and lyases. Whilst much is known about the structure and function of the hydrolases, relatively little is known about the structures and mechanisms of the corresponding lyases. To this end, crystals of the catalytic module of a novel family 10 pectate lyase, Pel10A from Pseudomonas cellulosa, were obtained using polyethylene glycol 2000 monomethylether as a precipitant. They belong to space group P2(1), with unit-cell parameters a = 47.7, b = 106.1, c = 55.4 A, beta = 92.0 degrees, and have two molecules in the asymmetric unit. The crystals diffract beyond 1.5 A using synchrotron radiation.

Influence of the aglycone region of the substrate binding cleft of Pseudomonas xylanase 10A on catalysis.

Pseudomonas cellulosa xylanase 10A (Pc Xyn10A) contains an extended substrate binding cleft comprising three glycone (-1 to -3) and four aglycone (+1 to +4) subsites and, typical of retaining glycoside hydrolases, exhibits transglycosylation activity at elevated substrate concentrations. In a previous study [Charnock, S. J., et al. (1997) J. Biol. Chem. 272, 2942-2951], it was demonstrated that the -2 subsite mutations E43A and N44A caused a 100-fold reduction in activity against xylooligosaccharides, but did not influence xylanase activity. This led to the proposal that the low activity of these mutants against xylooligosaccharides was due to nonproductive complex formation between these small substrates and the extended aglycone region of the active site. To test this hypothesis, key residues at the +2 (Asn182), +3 (Tyr255), and +4 (Tyr220) subsites were substituted for alanine, and the activity of the mutants against polysaccharides and oligosaccharides was evaluated. All the aglycone mutants exhibited greatly reduced or no transglycosylating activity, and the triple mutants, E43A/Y220A/Y255A and E43A/N182A/Y255A, had activity against xylotriose similar to that of E43A. The aglycone mutations caused an increase in both k(cat) and K(m) against xylan, with N182A/Y220A/Y255A and N182A/Y255A exhibiting 25- and 15-fold higher k(cat) values, respectively, than wild-type Pc Xyn10A. These data indicate that Glu43 plays a role in binding xylooligosaccharides, but not xylan, suggesting that the mechanisms by which Pc Xyn10A binds polysaccharides and oligosaccharides are distinct. The increased k(cat) of the mutants against xylan indicates that the aglycone region of wild-type Pc Xyn10A restricts the rate of catalysis by limiting diffusion of the cleaved substrate, generated at the completion of the k(2) step, out of the active site.

Pectate lyase 10A from Pseudomonas cellulosa is a modular enzyme containing a family 2a carbohydrate-binding module.

Pectate lyase 10A (Pel10A) enzyme from Pseudomonas cellulosa is composed of 649 residues and has a molecular mass of 68.5 kDa. Sequence analysis revealed that Pel10A contained a signal peptide and two serine-rich linker sequences that separate three modules. Sequence similarity was seen between the 9.2 kDa N-terminal module of Pel10A and family 2a carbohydrate-binding modules (CBMs). This N-terminal module of Pel10A was shown to encode an independently functional module with affinity to crystalline cellulose. A high sequence identity of 66% was seen between the 14.2 kDa central module of Pel10A and the functionally uncharacterized central modules of the xylan-degrading enzymes endoxylanase 10B, arabinofuranosidase 62C and esterase 1D, also from P. cellulosa. The 35.8 kDa C-terminal module of Pel10A was shown to have 30 and 36% identities with the family 10 pectate lyases from Azospirillum irakense and an alkaliphilic strain of Bacillus sp. strain KSM-P15, respectively. This His-tagged C-terminal module of the Pel10A was shown to encode an independent catalytic module (Pel10Acm). Pel10Acm was shown to cleave pectate and pectin in an endo-fashion and to have optimal activity at pH 10 and in the presence of 2 mM Ca2+. Highest enzyme activity was detected at 62 degrees C. Pel10Acm was shown to be most active against pectate (i.e. polygalacturonic acid) with progressively less activity against 31, 67 and 89% esterified citrus pectins. These data suggest that Pel10A has a preference for sequences of non-esterified galacturonic acid residues. Significantly, Pel10A and the P. cellulosa rhamnogalacturonan lyase 11A, in the accompanying article [McKie, Vincken, Voragen, van den Broek, Stimson and Gilbert (2001) Biochem. J. 355, 167-177], are the first CBM-containing pectinases described to date.

Substrate specificity in glycoside hydrolase family 10. Structural and kinetic analysis of the Streptomyces lividans xylanase 10A.

Endoxylanases are a group of enzymes that hydrolyze the beta-1, 4-linked xylose backbone of xylans. They are predominantly found in two discrete sequence families known as glycoside hydrolase families 10 and 11. The Streptomyces lividans xylanase Xyl10A is a family 10 enzyme, the native structure of which has previously been determined by x-ray crystallography at a 2.6 A resolution (Derewenda, U., Swenson, L., Green, R., Wei, Y., Morosoli, R., Shareck, F., Kluepfel, D., and Derewenda, Z. S. (1994) J. Biol. Chem. 269, 20811-20814). Here, we report the native structure of Xyl10A refined at a resolution of 1.2 A, which reveals many features such as the rare occurrence of a discretely disordered disulfide bond between residues Cys-168 and Cys-201. In order to investigate substrate binding and specificity in glycoside hydrolase family 10, the covalent xylobiosyl enzyme and the covalent cellobiosyl enzyme intermediates of Xyl10A were trapped through the use of appropriate 2-fluoroglycosides. The alpha-linked intermediate with the nucleophile, Glu-236, is in a (4)C(1) chair conformation as previously observed in the family 10 enzyme Cex from Cellulomonas fimi (Notenboom, V., Birsan, C., Warren, R. A. J., Withers, S. G., and Rose, D. R. (1998) Biochemistry 37, 4751-4758). The different interactions of Xyl10A with the xylobiosyl and cellobiosyl moieties, notably conformational changes in the -2 and -1 subsites, together with the observed kinetics on a range of aryl glycosides, shed new light on substrate specificity in glycoside hydrolase family 10.

The X6 "thermostabilizing" domains of xylanases are carbohydrate-binding modules: structure and biochemistry of the Clostridium thermocellum X6b domain.

Many polysaccharide-degrading enzymes display a modular structure in which a catalytic module is attached to one or more noncatalytic modules. Several xylanases contain a module of previously unknown function (termed "X6" modules) that had been implicated in thermostability. We have investigated the properties of two such "thermostabilizing" modules, X6a and X6b from the Clostridium thermocellumxylanase Xyn10B. These modules, expressed either as discrete entities or as their natural fusions with the catalytic module, were assayed, and their capacity to bind various carbohydrates and potentiate hydrolytic activity was determined. The data showed that X6b, but not X6a, increased the activity of the enzyme against insoluble xylan and bound specifically to xylooligosaccharides and various xylans. In contrast, X6a exhibited no affinity for soluble or insoluble forms of xylan. Isothermal titration calorimetry revealed that the ligand-binding site of X6b accommodates approximately four xylose residues. The protein exhibited K(d) values in the low micromolar range for xylotetraose, xylopentaose, and xylohexaose; 24 microM for xylotriose; and 50 microM for xylobiose. Negative DeltaH and DeltaS values indicate that the interaction of X6b with xylooligosaccharides and xylan is driven by enthalpic forces. The three-dimensional structure of X6b has been solved by X-ray crystallography to a resolution of 2.1 A. The protein is a beta-sandwich that presents a tryptophan and two tyrosine residues on the walls of a shallow cleft that is likely to be the xylan-binding site. In view of the structural and carbohydrate-binding properties of X6b, it is proposed that this and related modules be re-assigned as family 22 carbohydrate-binding modules.

Substrate specificity in glycoside hydrolase family 10. Tyrosine 87 and leucine 314 play a pivotal role in discriminating between glucose and xylose binding in the proximal active site of Pseudomonas cellulosa xylanase 10A.

The Pseudomonas family 10 xylanase, Xyl10A, hydrolyzes beta1, 4-linked xylans but exhibits very low activity against aryl-beta-cellobiosides. The family 10 enzyme, Cex, from Cellulomonas fimi, hydrolyzes aryl-beta-cellobiosides more efficiently than does Xyl10A, and the movements of two residues in the -1 and -2 subsites are implicated in this relaxed substrate specificity (Notenboom, V., Birsan, C., Warren, R. A. J., Withers, S. G., and Rose, D. R. (1998) Biochemistry 37, 4751-4758). The three-dimensional structure of Xyl10A suggests that Tyr-87 reduces the affinity of the enzyme for glucose-derived substrates by steric hindrance with the C6-OH in the -2 subsite of the enzyme. Furthermore, Leu-314 impedes the movement of Trp-313 that is necessary to accommodate glucose-derived substrates in the -1 subsite. We have evaluated the catalytic activities of the mutants Y87A, Y87F, L314A, L314A/Y87F, and W313A of Xyl10A. Mutations to Tyr-87 increased and decreased the catalytic efficiency against 4-nitrophenyl-beta-cellobioside and 4-nitrophenyl-beta-xylobioside, respectively. The L314A mutation caused a 200-fold decrease in 4-nitrophenyl-beta-xylobioside activity but did not significantly reduce 4-nitrophenyl-beta-cellobioside hydrolysis. The mutation L314A/Y87A gave a 6500-fold improvement in the hydrolysis of glucose-derived substrates compared with xylose-derived equivalents. These data show that substantial improvements in the ability of Xyl10A to accommodate the C6-OH of glucose-derived substrates are achieved when steric hindrance is removed.

Structure of the nucleotide-diphospho-sugar transferase, SpsA from Bacillus subtilis, in native and nucleotide-complexed forms.

The enzymatic formation of glycosidic bonds may be catalyzed by the transfer of the glycosyl moiety from an activated nucleotide-diphospho-sugar donor to a specific acceptor. SpsA is a glycosyltransferase implicated in the synthesis of the spore coat of Bacillus subtilis, whose homologues include cellulose synthase and many lipopolysaccharide and bacterial O-antigen synthases. The three-dimensional crystal structure of SpsA has been determined by conventional MIR techniques at a resolution of 1.5 A. It is a two-domain protein with a nucleotide-binding domain together with an acceptor binding domain which features a disordered loop spanning the active site. The structures of SpsA in complex with both Mg-UDP and Mn-UDP have also been determined at 2.0 and 1.7 A, respectively. These complexes, together with the sequence conservation, begin to shed light on the mechanism of this ubiquitous family of inverting glycosyltransferases.

Cloning, crystallization and preliminary X-ray analysis of a nucleotide-diphospho-sugar transferase spsA from Bacillus subtilis.

Nucleotide-diphospho-sugar transferases represent, in terms of quantity, one of the most important groups of enzymes on Earth, yet little is known about their structure and mechanism. Such a transferase, the spsA gene product involved in the synthesis of the bacterial spore coat in Bacillus subtilis, has been cloned and over-expressed in an Escherichia coli expression system. Crystals have been grown, using PEG 8000 as a precipitant, in a form suitable for high-resolution X-ray analysis. They belong to space group C2221, with unit-cell dimensions a = 42.4, b = 142.0, c = 81.4 A and with one molecule of spsA in the asymmetric unit. The crystals diffract beyond 1.5 A using synchrotron radiation.

The topology of the substrate binding clefts of glycosyl hydrolase family 10 xylanases are not conserved.

The crystal structures of family 10 xylanases indicate that the distal regions of their active sites are quite different, suggesting that the topology of the substrate binding clefts of these enzymes may vary. To test this hypothesis, we have investigated the rate and pattern of xylooligosaccharide cleavage by the family 10 enzymes, Pseudomonas fluorescens subsp. cellulosa xylanase A (XYLA) and Cellulomonas fimi exoglucanase, Cex. The data showed that Cex contained three glycone and two aglycone binding sites, while XYLA had three glycone and four aglycone binding sites, supporting the view that the topologies of substrate binding clefts in family 10 glycanases are not highly conserved. The importance of residues in the substrate binding cleft of XYLA in catalysis and ligand binding were evaluated using site-directed mutagenesis. In addition to providing insight into the function of residues in the glycone region of the active site, the data showed that the aromatic residues Phe-181, Tyr-255, and Tyr-220 play important roles in binding xylose moieties, via hydrophobic interactions, at subsites +1, +3, and +4, respectively. Interestingly, the F181A mutation caused a much larger reduction in the activity of the enzyme against xylooligosaccharides compared with xylan. These data, in conjunction with a previous study (Charnock, S. J., Lakey, J. H., Virden, R., Hughes, N., Sinnott, M. L., Hazlewood, G. P., Pickersgill, R., and Gilbert, H. J. (1997) J. Biol. Chem. 272, 2942-2951), suggest that the binding of xylooligosaccharides at the -2 and +1 subsites ensures that the substrates occupy the -1 and +1 subsites and thus preferentially form productive complexes with the enzyme. Loss of ligand binding at either subsite results in small substrates forming nonproductive complexes with XYLA by binding to distal regions of the substrate binding cleft.

Key residues in subsite F play a critical role in the activity of Pseudomonas fluorescens subspecies cellulosa xylanase A against xylooligosaccharides but not against highly polymeric substrates such as xylan.

In a previous study crystals of Pseudomonas fluorescens subspecies cellulosa xylanase A (XYLA) containing xylopentaose revealed that the terminal nonreducing end glycosidic bond of the oligosaccharide was adjacent to the catalytic residues of the enzyme, suggesting that the xylanase may have an exo-mode of action. However, a cluster of conserved residues in the substrate binding cleft indicated the presence of an additional subsite, designated subsite F. Analysis of the biochemical properties of XYLA revealed that the enzyme was a typical endo-beta1,4-xylanase, providing support for the existence of subsite F. The three-dimensional structure of four family 10 xylanases, including XYLA, revealed several highly conserved residues that are on the surface of the active site cleft. To investigate the role of some of these residues, appropriate mutations of XYLA were constructed, and the biochemical properties of the mutated enzymes were evaluated. N182A hydrolyzed xylotetraose to approximately equal molar quantities of xylotriose, xylobiose, and xylose, while native XYLA cleaved the substrate to primarily xylobiose. These data suggest that N182 is located at the C site of the enzyme. N126A and K47A were less active against xylan and aryl-beta-glycosides than native XYLA. The potential roles of Asn-126 and Lys-47 in the function of the catalytic residues are discussed. E43A and N44A, which are located in the F subsite of XYLA, retained full activity against xylan but were significantly less active than the native enzyme against oligosaccharides smaller than xyloseptaose. These data suggest that the primary role of the F subsite of XYLA is to prevent small oligosaccharides from forming nonproductive enzyme-substrate complexes.

Evidence that galactanase A from Pseudomonas fluorescens subspecies cellulosa is a retaining family 53 glycosyl hydrolase in which E161 and E270 are the catalytic residues.

A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA was screened for galactanase-positive recombinants. The nine galactanase positive phage isolated contained the same galactanase gene designated galA. The deduced primary structure of the enzyme (galactanase A; GalA) encoded by galA had a Mr of 42 130 and exhibited significant sequence identity with a galactanase from Aspergillus aculeatus, placing GalA in glycosyl hydrolase family 53. The enzyme displayed properties typical of an endo-beta1, 4-galactanase and exhibited no activity against the other plant structural polysaccharides evaluated. Analysis of the stereochemical course of 2,4-dinitrophenyl-beta-galactobioside (2,4-DNPG2) hydrolysis by GalA indicated that the galactanase catalyzes the hydrolysis of glycosidic bonds by a double displacement general acid-base mechanism. Hydrophobic cluster analysis (HCA) suggested that family 53 enzymes are related to the GH-A clan of glycosyl hydrolases, which have an (alpha/beta)8 barrel structure. HCA also predicted that E161 and E270 were the acid-base and nucleophilic residues, respectively. Mutants of GalA in which E161 and E270 had been replaced with alanine residues were essentially inactive against galactan. Against 2,4-DNPG2, E161A exhibited a much lower Km and kcat than native GalA, while E270A was inactive against the substrate. Analysis of the pre-steady-state kinetics of 2,4-DNPG2 hydrolysis by E161A showed that there was an initial rapid release of 2,4-dinitrophenol (2,4-DNP), which then decayed to a slow steady-state rate of product formation. No pre-steady-state burst of 2,4-DNP release was observed with the wild-type enzyme. These data are consistent with the HCA prediction that E161 and E270 are the acid-base and nucleophilic catalytic residues of GalA, respectively.