Calcium-dependent catalytic activity of a novel phytase from Bacillus amyloliquefaciens DS11

The thermostable phytase from Bacillus amyloliquefaciens DS11 hydrolyzes phytate (myo-inositol hexakisphosphate, IP6) to less phosphorylated myo-inositol phosphates in the presence of Ca2+. In this report, we discuss the unique Ca2+-dependent catalytic properties of the phytase and its specific substrate requirement. Initial rate kinetic studies of the phytase indicate that the enzyme activity follows a rapid equilibrium ordered mechanism in which binding of Ca2+ to the active site is necessary for the essential activation of the enzyme. Ca2+ turned out to be also required for the substrate because the phytase is only able to hydrolyze the calcium-phytate complex. In fact, both an excess amount of free Ca2+ and an excess of free phytate, which is not complexed with each other, can act as competitive inhibitors. The Ca2+-dependent catalytic activity of the enzyme was further confirmed, and the critical amino acid residues for the binding of Ca2+ and substrate were identified by site-specific mutagenesis studies. Isothermal titration calorimetry (ITC) was used to understand if the decreased enzymatic activity was related to poor Ca2+ binding. The pH dependence of the Vmax and Vmax/Km consistently supported these observations by demonstrating that the enzyme activity is dependent on the ionization of amino acid residues that are important for the binding of Ca2+ and the substrate. The Ca2+-dependent activation of enzyme and substrate was found to be different from other histidine acid phytases that hydrolyze metal-free phytate.     

Site-directed mutagenesis of an alkaline phytase: influencing specificity, activity and stability in acidic milieu

Site-directed mutagenesis of a thermostable alkaline phytase from Bacillus sp. MD2 was performed with an aim to increase its specific activity and activity and stability in an acidic environment. The mutation sites are distributed on the catalytic surface of the enzyme (P257R, E180N, E229V and S283R) and in the active site (K77R, K179R and E227S). Selection of the residues was based on the idea that acid active phytases are more positively charged around their catalytic surfaces. Thus, a decrease in the content of negatively charged residues or an increase in the positive charges in the catalytic region of an alkaline phytase was assumed to influence the enzyme activity and stability at low pH. Moreover, widening of the substrate-binding pocket is expected to improve the hydrolysis of substrates that are not efficiently hydrolysed by wild type alkaline phytase. Analysis of the phytase variants revealed that E229V and S283R mutants increased the specific activity by about 19% and 13%, respectively. Mutation of the active site residues K77R and K179R led to severe reduction in the specific activity of the enzyme. Analysis of the phytase mutant-phytate complexes revealed increase in hydrogen bonding between the enzyme and the substrate, which might retard the release of the product, resulting in decreased activity. On the other hand, the double mutant (K77R-K179R) phytase showed higher stability at low pH (pH 2.6-3.0). The E227S variant was optimally active at pH 5.5 (in contrast to the wild type enzyme that had an optimum pH of 6) and it exhibited higher stability in acidic condition. This mutant phytase, displayed over 80% of its initial activity after 3 h incubation at pH 2.6 while the wild type phytase retained only about 40% of its original activity. Moreover, the relative activity of this mutant phytase on calcium phytate, sodium pyrophosphate and p-nitro phenyl phosphate was higher than that of the wild type phytase.     

Semi-rational site-directed mutagenesis of phyI1s from <Emphasis Type="Italic">Aspergillus niger</Emphasis> 113 at two residue to improve its phytase activity

Through alignment of amino acid sequences among different phytases, we found that the amino acid at residues 53 and 91 vary broadly. To prove that the amino acid at residues 53 and 91 were related to phytase specific activity, two single mutant phyI1s Q53R and K91D were obtained by site-directed mutagenesis strategy. None of the single amino acid residues in the two mutants was in a position reported to be important for catalysis or substrate binding. Kinetic analysis of the phytase activity of the two mutants (Q53R and K91D) indicated that the mutants were attributed to 2.2- and 1.5-fold increased specific activity, and a 1.47- and 1.16-fold increased affinity for sodium phytate. In addition, the overall catalytic efficiency (k _cat/K _m) of the two mutants was improved 4.08- and 2.84-fold compared to that of the wild type. Such mutants will be instrumental for the structure–function study of the enzyme and for industrial application.     

Crystal structures of Bacillus alkaline phytase in complex with divalent metal ions and inositol hexasulfate

Alkaline phytases from Bacillus species, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives to animal feed. The thermostability and neutral optimum pH of Bacillus phytase are attributed largely to the presence of calcium ions. Nonetheless, no report has demonstrated directly how the metal ions coordinate phytase and its substrate to facilitate the catalytic reaction. In this study, the interactions between a phytate analog (myo-inositol hexasulfate) and divalent metal ions in Bacillus subtilis phytase were revealed by the crystal structure at 1.25 Å resolution. We found all, except the first, sulfates on the substrate analog have direct or indirect interactions with amino acid residues in the enzyme active site. The structures also unraveled two active site-associated metal ions that were not explored in earlier studies. Significantly, one metal ion could be crucial to substrate binding. In addition, binding of the fourth sulfate of the substrate analog to the active site appears to be stronger than that of the others. These results indicate that alkaline phytase starts by cleaving the fourth phosphate, instead of the third or the sixth that were proposed earlier. Our high-resolution, structural representation of Bacillus phytase in complex with a substrate analog and divalent metal ions provides new insight into the catalytic mechanism of alkaline phytases in general.     

Site-directed mutagenesis of phosphate-contacting amino acids of bovine pancreatic deoxyribonuclease I

Bovine pancreatic deoxyribonuclease I (DNase I) is an endonuclease which cleaves double-stranded DNA. Cocrystal structures of DNase I with oligonucleotides have revealed interactions between the side chains of several amino acids (N74, R111, N170, S206, T207, and Y211) and the DNA phosphates. The effects these interactions have on enzyme catalysis and DNA hydrolysis selectivity have been investigated by site-directed mutagenesis. Mutations to R111, N170, T207, and Y211 severely compromised activity toward both DNA and a small chromophoric substrate. A hydrogen bond between R111 (which interacts with the phosphate immediately 5' to the cutting site) and the essential amino acid H134 is probably required to maintain this histidine in the correct orientation for efficient hydrolysis. Both T207 and Y211 bind to the phosphate immediately 3' to the cleavage site. Additionally, T207 is involved in binding an essential, structural, calcium ion, and Y211 is the nearest neighbor to D212, a critical catalytic residue. N170 interacts with the scissile phosphate and appears to play a direct role in the catalytic mechanism. The mutation N74D, which interacts with a phosphate twice removed from the scissile group, strongly reduced DNA hydrolysis. However, a comparison of DNase I variants from several species suggests that certain amino acids, which allow interaction with phosphates (positively charged or hydrogen bonding), are tolerated. S206, which binds to a DNA phosphate two positions away from the cleavage site, appears to play a relatively unimportant role. None of the enzyme variants, including a triple mutation in which N74, R111, and Y211 were altered, affected DNA hydrolysis selectivity. This suggests that phosphate binding residues play no role in the selection of DNA substrates.     

Site-directed mutagenesis of Aspergillus niger NRRL 3135 phytase at residue 300 to enhance catalysis at pH 4.0

Increased phytase activity for Aspergillus niger NRRL 3135 phytaseA (phyA) at intermediate pH levels (3.0-5.0) was achieved by site-directed mutagenesis of its gene at amino acid residue 300. A single mutation, K300E, resulted in an increase of the hydrolysis of phytic acid of 56% and 19% at pH 4.0 and 5.0, respectively, at 37 degrees C. This amino acid residue has previously been identified as part of the substrate specificity site for phyA and a comparison of the amino acid sequences of other cloned fungal phytases indicated a correlation between a charged residue at this position and high specific activity for phytic acid hydrolysis. The substitution at this residue by either another basic (R), uncharged (T), or acidic amino acid (D) did not yield a recombinant enzyme with the same favorable properties. Therefore, we conclude that this residue is not only important for the catalytic function of phyA, but also essential for imparting a favorable pH environment for catalysis.     

Avian multiple inositol polyphosphate phosphatase is an active phytase that can be engineered to help ameliorate the planet's "phosphate crisis"

Contemporary phytase research is primarily concerned with ameliorating the problem of inadequate digestion of inositol hexakisphosphate (phytate; InsP6) in monogastric farm animal feed, so as to reduce the pollution that results from the high phosphate content of the manure. In the current study we pursue a new, safe and cost-effective solution. We demonstrate that the rate of hydrolysis of InsP6 by recombinant avian MINPP (0.7 micromol/mg protein/min) defines it as by far the most active phytase found to date in any animal cell (the corresponding activity of recombinant mammalian MINPP is only 0.006 micromol/mg protein/min). Although avian MINPP has less than 20% sequence identity with microbial phytases, we create a homology model of MINPP in which it is predicted that the structure of the phytase active site is well-conserved. This model is validated by site-directed mutagenesis and by use of a substrate analogue, scyllo-InsP6, which we demonstrate is only a weak MINPP substrate. In a model chicken cell line, we overexpressed a mutant form of MINPP that is secretion-competent. This version of the enzyme was actively secreted without affecting either cell viability or the cellular levels of any inositol phosphates. Our studies offer a genetic strategy for greatly improving dietary InsP6 digestion in poultry.     

Structures of Selenomonas ruminantium phytase in complex with persulfated phytate: DSP phytase fold and mechanism for sequential substrate hydrolysis

Various inositide phosphatases participate in the regulation of inositol polyphosphate signaling molecules. Plant phytases are phosphatases that hydrolyze phytate to less-phosphorylated myo-inositol derivatives and phosphate. The phytase from Selenomonas ruminantium shares no sequence homology with other microbial phytases. Its crystal structure revealed a phytase fold of the dual-specificity phosphatase type. The active site is located near a conserved cysteine-containing (Cys241) P loop. We also solved two other crystal forms in which an inhibitor, myo-inositol hexasulfate, is cocrystallized with the enzyme. In the "standby" and the "inhibited" crystal forms, the inhibitor is bound, respectively, in a pocket slightly away from Cys241 and at the substrate binding site where the phosphate group to be hydrolyzed is held close to the -SH group of Cys241. Our structural and mutagenesis studies allow us to visualize the way in which the P loop-containing phytase attracts and hydrolyzes the substrate (phytate) sequentially.     

Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase.

Using site-directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme-substrate complex to 108 A resolution using a catalytically incompetent mutant (E172C). In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme. On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid-base catalyst in the reaction.     

Degradation of phytate in the gut of pigs ‐ pathway of gastrointestinal inositol phosphate hydrolysis and enzymes involved

The present study gives an overview on the whole mechanism of phytate degradation in the gut and the enzymes involved. Based on the similarity of the human and pigs gut, the study was carried out in pigs as model for humans. To differentiate between intrinsic feed phytases and endogenous phytases hydrolysing phytate in the gut, two diets, one high (control diet) and the other one very low in intrinsic feed phytases (phytase inactivated diet) were applied. In the chyme of stomach, small intestine and colon inositol phosphate isomers and activities of phytases and alkaline phosphatases were determined. In parallel total tract phytate degradation and apparent phosphorus digestibility were assessed. In the stomach chyme of pigs fed the control diet, comparable high phytase activity and strong phytate degradation were observed. The predominant phytate hydrolysis products were inositol phosphates, typically formed by plant phytases. For the phytase inactivated diet, comparable very low phytase activity and almost no phytate degradation in the stomach were determined. In the small intestine and colon, high activity of alkaline phosphatases and low activity of phytases were observed, irrespective of the diet fed. In the colon, stronger phytate degradation for the phytase inactivated diet than for the control diet was detected. Phytate degradation throughout the whole gut was nearly complete and very similar for both diets while the apparent availability of total phosphorus was significantly higher for the pigs fed the control diet than the phytase inactivated diet. The pathway of inositol phosphate hydrolysis in the gut has been elucidated.     

A novel extracellular low‐temperature active phytase from Bacillus aryabhattai RS1 with potential application in plant growth

Bacillus aryabhattai RS1 isolated from rhizosphere produced an extracellular, low temperature active phytase. The cultural conditions for enzyme production were optimized to obtain 35 U mL^?1 of activity. Purified phytase had specific activity and molecular weight of 72.97 U mg^?1 and ～40 kDa, respectively. The enzyme was optimally active at pH 6.5 and 40°C and was highly specific to phytate. It exhibited higher catalytic activity at low temperature, retaining over 40% activity at 10°C. Phytase was more thermostable in presence of Ca²⁺ ion and retained 100% residual activity on preincubation at 20–50°C for 30 min. Partial phytase encoding gene, phy_B (816 bp) was cloned and sequenced. The encoded amino acid sequence (272 aa) contained two conserved motifs, DA[A/T/E]DDPA[I/L/V]W and NN[V/I]D[I/L/V]R[Y/D/Q] of β‐propellar phytase and had lower sequence homology with other Bacillus phytases, indicating its novelty. Phytase and the bacterial inoculum were effective in improving germination and growth of chickpea seedlings under phosphate limiting condition. Moreover, the potential applications of the enzyme with relatively high activity at lower temperatures (20–30°C) could also be extended to aquaculture and food processing. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:633–641, 2017     

The tandemly repeated domains of a β-propeller phytase act synergistically to increase catalytic efficiency

β-Propeller phytases (BPPs) with tandemly repeated domains are abundant in nature. Previous studies have shown that the intact domain is responsible for phytate hydrolysis, but the function of the other domain is relatively unknown. In this study, a new dual-domain BPP (PhyH) from Bacillus sp. HJB17 was identified to contain an incomplete N-terminal BPP domain (PhyH-DI, residues 41-318) and a typical BPP domain (PhyH-DII, residues 319-644) at the C-terminus. Purified recombinant PhyH and PhyH-DII required Ca(2+) for phytase activity, showed activity at low temperatures (0-35 °C) and pH 6.0-8.0, and remained active (at 37 °C) after incubation at 60 °C and pH 6.0-12.0. Compared with PhyH-DII, PhyH is catalytically more active against phytate (catalytic constant 27.72 versus 4.17 s(-1)), which indicates the importance of PhyH-DI in phytate degradation. PhyH-DI was found to hydrolyze phytate intermediate D-Ins(1,4,5,6) P(4), and to act synergistically (a 1.2-2.5-fold increase in phosphate release) with PhyH-DII, other BPPs (PhyP and 168PhyA) and a histidine acid phosphatase. Furthermore, fusion of PhyH-DI with PhyP or 168PhyA significantly enhanced their catalytic efficiencies. This is the first report to elucidate the substrate specificity of the incomplete domain and the functional relationship of tandemly repeated domains in BPPs. We conjecture that dual-domain BPPs have succeeded evolutionarily because they can increase the amount of available phosphate by interacting together. Additionally, fusing PhyH-DI to a single-domain phytase appears to be an efficient way to improve the activity of the latter.     

Pathway of phytate dephosphorylation by beta-propeller phytases of different origins

Using a combination of high-performance ion chromatography analysis and kinetic studies, the pathway of myo-inositol hexakisphosphate dephosphorylation by the beta-propeller phytase of Shewanella oneidensis was established, which was then compared with that of Bacillus subtilis 168, Bacillus amyloliquefaciens ATCC 15841, and B. amyloliquefaciens 45 beta-propeller phytases. The data demonstrate that all of these beta-propeller phytases dephosphorylate myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via d-Ins(1,2,4,5,6)P5, Ins(2,4,5,6)P4 to finally Ins(2,4,6)P3. Thus, the beta-propeller phytases prefer the hydrolysis of every second phosphate over that of adjacent ones. This finding does not support previous phytate degradation models proposed by J. Kerovuo, J. Rouvinen, and F. Hatzack (2000. Biochem. J. 352: 623-628) and R. Greiner, A. Farouk, M. Larsson Alminger, and N.G. Carlsson (2002. Can. J. Microbiol. 48: 986-994), but seems to fit with the structural model given by S. Shin, N.C. Ha, B.C. Oh, T.K. Oh, and B.H. Oh (2001. Structure, 9: 851-858).     

Enzyme mechanism and catalytic property of beta propeller phytase

BACKGROUND: Phytases hydrolyze phytic acid (myo-inositol-hexakisphosphate) to less-phosphorylated myo-inositol derivatives and inorganic phosphate. Phytases are used in animal feed to reduce phosphate pollution in the environment. Recently, a thermostable, calcium-dependent Bacillus phytase was identified that represents the first example of the beta propeller fold exhibiting phosphatase activity. We sought to delineate the catalytic mechanism and property of this enzyme. RESULTS: The crystal structure of the enzyme in complex with inorganic phosphate reveals that two phosphates and four calcium ions are tightly bound at the active site. Mutation of the residues involved in the calcium chelation results in severe defects in the enzyme's activity. One phosphate ion, chelating all of the four calcium ions, is close to a water molecule bridging two of the bound calcium ions. Fluoride ion, which is expected to replace this water molecule, is an uncompetitive inhibitor of the enzyme. The enzyme is able to hydrolyze any of the six phosphate groups of phytate. CONCLUSIONS: The enzyme reaction is likely to proceed through a direct attack of the metal-bridging water molecule on the phosphorous atom of a substrate and the subsequent stabilization of the pentavalent transition state by the bound calcium ions. The enzyme has two phosphate binding sites, the "cleavage site", which is responsible for the hydrolysis of a substrate, and the "affinity site", which increases the binding affinity for substrates containing adjacent phosphate groups. The existence of the two nonequivalent phosphate binding sites explains the puzzling formation of the alternately dephosphorylated myo-inositol triphosphates from phytate and the hydrolysis of myo-inositol monophosphates.     

Isolation, Characterization, Molecular Gene Cloning, and Sequencing of a Novel Phytase from Bacillus subtilis

The Bacillus subtilis strain VTT E-68013 was chosen for purification and characterization of its excreted phytase. Purified enzyme had maximal phytase activity at pH 7 and 55°C. Isolated enzyme required calcium for its activity and/or stability and was readily inhibited by EDTA. The enzyme proved to be highly specific since, of the substrates tested, only phytate, ADP, and ATP were hydrolyzed (100, 75, and 50% of the relative activity, respectively). The phytase gene (phyC) was cloned from the B. subtilis VTT E-68013 genomic library. The deduced amino acid sequence (383 residues) showed no homology to the sequences of other phytases nor to those of any known phosphatases. PhyC did not have the conserved RHGXRXP sequence found in the active site of known phytases, and therefore PhyC appears not to be a member of the phytase subfamily of histidine acid phosphatases but a novel enzyme having phytase activity. Due to its pH profile and optimum, it could be an interesting candidate for feed applications.     

Molecular and physiological characterisation of a 3-phytase from soil bacterium<Emphasis Type="Italic"> Klebsiella</Emphasis> sp. ASR1

Klebsiella sp. strain ASR1 isolated from an Indonesian rice field is able to hydrolyse myo-inositol hexakis phosphate (phytate). The phytase protein was purified and characterised as a 42 kDa protein accepting phytate, NADP and sugar phosphates as substrates. The corresponding gene (phyK) was cloned from chromosomal DNA using a combined approach of protein and genome analysis, and expressed in Escherichia coli. The recombinant enzyme was identified as a 3-phytase yielding myo-inositol monophosphate, Ins(2)P, as the final product of enzymatic phytate hydrolysis. Based on its amino acid sequence, PhyK appears to be a member of a hitherto unknown subfamily of histidine acid phytate-degrading enzymes with the active site RHGXRXP and HD sequence motifs, and is different from other general phosphatases and phytases. Due to its ability to degrade sodium phytate to the mono phosphate ester, the phyK gene product is an interesting candidate for industrial and agricultural applications to make phytate phosphorous available for plant and animal nutrition.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Engineering E. coli Alkaline Phosphatase Yields Changes of Catalytic Activity,Thermal Stability and Phosphate Inhibition

To investigate the function of aspartic acid residue 101 and arginine residue 166 in the active site of Escherichia coli alkaline phosphatase (EAP), two single mutants D101S (Asp 101 &#77 Ser) and R166K (Arg 166 &#77 Lys) and a double mutant D101S/R166K of EAP were generated through site-directed mutagenesis based on over-lap PCR method. Their enzymatic kinetic properties, thermal stabilities and possible reaction mechanism were explored. In the presence of inorganic phosphate acceptor, 1 M diethanolamine buffer, the k cat for D101S mutant enzyme increased 10-fold compared to that of wild-type EAP. The mutant R166K has a 2-fold decrease of k cat relative to the wild-type EAP, but the double mutant D101S/R166K was in the middle of them, indicative of an additive effect of these two mutations. On the other hand, the catalytic efficiencies of mutant enzymes are all reduced because of a substantial increase of K m values. All three mutants were more resistant to phosphate inhibitor than the wild-type enzyme. The analysis of the kinetic data suggests that (1) the D101S mutant enzyme obtains a higher catalytic activity by allowing a faster release of the product; (2) the R166K mutant enzyme can reduce the binding of the substrate and phosphate competitive inhibitor; (3) the double mutant enzyme has characteristics of both quicker catalytic turnover number and decreased affinity for competitive inhibitor. Additionally, pre-steady-state kinetics of D101S and D101S/R166K mutants revealed a transient burst followed by a linear steady state phase, obviously different from that of wild-type EAP, suggesting that the rate-limiting step has partially change from the release of phosphate from non-covalent E-Pi complex to the hydrolysis of covalent E-Pi complex for these two mutants.     

Biochemical properties and substrate specificities of alkaline and histidine acid phytases

Phytases are a special class of phosphatase that catalyze the sequential hydrolysis of phytate to less-phosphorylated myo-inositol derivatives and inorganic phosphate. Phytases are added to animal feedstuff to reduce phosphate pollution in the environment, since monogastric animals such as pigs, poultry, and fish are unable to metabolize phytate. Based on biochemical properties and amino acid sequence alignment, phytases can be categorized into two major classes, the histidine acid phytases and the alkaline phytases. The histidine acid phosphatase class shows broad substrate specificity and hydrolyzes metal-free phytate at the acidic pH range and produces myo-inositol monophosphate as the final product. In contrast, the alkaline phytase class exhibits strict substrate specificity for the calcium–phytate complex and produces myo-inositol trisphosphate as the final product. This review describes recent findings that present novel viewpoints concerning the molecular basis of phytase classification.     

Molecular characterization,physicochemical properties,known and potential applications of phytases: An overview

Phytases (myo-inositol hexakisphosphate phosphohydrolases) hydrolyze the phosphate ester bonds of phytate-releasing phosphate and lower myo-inositol phosphates and/or myo-inositol. Phytases, in general, are known to enhance phosphate and mineral uptake in monogastric animals such as poultry, swine, and fish, which cannot metabolize phytate besides reducing environmental pollution significantly. In this study, the molecular, biophysical, and biochemical properties of phytases are reviewed in detail. Alterations in the molecular and catalytic properties of phytases, upon expression in heterologous hosts, are discussed. Diverse applications of phytases as feed additives, as soil amendment, in aquaculture, development of transgenic organisms, and as nutraceuticals in the human diet also are dealt with. Furthermore, phytases are envisaged to serve as potential enzymes that can produce versatile lower myo-inositol phosphates of pharmaceutical importance. Development of phytases with improved attributes is an important area being explored through genetic and protein engineering approaches, as no known phytase can fulfill all the properties of an ideal feed additive.     

Processivity, substrate binding, and mechanism of cellulose hydrolysis by Thermobifida fusca Cel9A

Thermobifida fusca Cel9A-90 is a processive endoglucanase consisting of a family 9 catalytic domain (CD), a family 3c cellulose binding module (CBM3c), a fibronectin III-like domain, and a family 2 CBM. This enzyme has the highest activity of any individual T. fusca enzyme on crystalline substrates, particularly bacterial cellulose (BC). Mutations were introduced into the CD or the CBM3c of Cel9A-68 using site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli; purified; and tested for activity on four substrates, ligand binding, and processivity. The results show that H125 and Y206 play an important role in activity by forming a hydrogen bonding network with the catalytic base, D58; another important supporting residue, D55; and Glc(-1) O1. R378, a residue interacting with Glc(+1), plays an important role in processivity. Several enzymes with mutations in the subsites Glc(-2) to Glc(-4) had less than 15% activity on BC and markedly reduced processivity. Mutant enzymes with severalfold-higher activity on carboxymethyl cellulose (CMC) were found in the subsites from Glc(-2) to Glc(-4). The CBM3c mutant enzymes, Y520A, R557A/E559A, and R563A, had decreased activity on BC but had wild-type or improved processivity. Mutation of D513, a conserved residue at the end of the CBM, increased activity on crystalline cellulose. Previous work showed that deletion of the CBM3c abolished crystalline activity and processivity. This study shows that it is residues in the catalytic cleft that control processivity while the CBM3c is important for loose binding of the enzyme to the crystalline cellulose substrate.