Structural Basis for Substrate Selectivity in Human Maltase-Glucoamylase and Sucrase-Isomaltase N-terminal Domains

Human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are small intestinal enzymes that work concurrently to hydrolyze the mixture of linear α-1,4- and branched α-1,6-oligosaccharide substrates that typically make up terminal starch digestion products. MGAM and SI are each composed of duplicated catalytic domains, N- and C-terminal, which display overlapping substrate specificities. The N-terminal catalytic domain of human MGAM (ntMGAM) has a preference for short linear α-1,4-oligosaccharides, whereas N-terminal SI (ntSI) has a broader specificity for both α-1,4- and α-1,6-oligosaccharides. Here we present the crystal structure of the human ntSI, in apo form to 3.2 Å and in complex with the inhibitor kotalanol to 2.15 Å resolution. Structural comparison with the previously solved structure of ntMGAM reveals key active site differences in ntSI, including a narrow hydrophobic +1 subsite, which may account for its additional substrate specificity for α-1,6 substrates.     

Structure-Guided Systems-Level Engineering of Oxidation-Prone Methionine Residues in Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica for Significant Improvement of Both Oxidative Stability and Catalytic Efficiency

High oxidative stability and catalytic efficiency are required for the alkaline α-amylases to keep the enzymatic performance under the harsh conditions in detergent industries. In this work, we attempted to significantly improve both the oxidative stability and catalytic efficiency of an alkaline α-amylase from Alkalimonas amylolytica by engineering the five oxidation-prone methionine residues around the catalytic domain via a systematic approach. Specifically, based on the tertiary structure analysis, five methionines (Met 145, Met 214, Met 229, Met 247 and Met 317) were individually substituted with oxidation-resistant threonine, isoleucine and alaline, respectively. Among the created 15 mutants, 7 mutants M145A, M145I, M214A, M229A, M229T, M247T and M317I showed significantly enhanced oxidative stability or catalytic efficiency. In previous work, we found that the replacement of M247 with leucine could significantly improve the oxidative stability. Thus, these 8 positive mutants (M145A, M145I, M214A, M229A, M229T, M247T, M247L and M317I) were used to conduct the second round of combinational mutations. Among the constructed 85 mutants (25 two-point mutants, 36 three-point mutants, 16 four-point mutants and 8 five-point mutants), the mutant M145I-214A-229T-247T-317I showed a 5.4-fold increase in oxidative stability and a 3.0-fold increase in catalytic efficiency. Interestingly, the specific activity, alkaline stability and thermal stability of this mutant were also increased. The increase of salt bridge and hydrogen bonds around the catalytic domain contributed to the significantly improved catalytic efficiency and stability, as revealed by the three-dimensional structure model of wild-type alkaline α-amylase and its mutant M145I-214A-229T-247T-317I. With the significantly improved oxidative stability and catalytic efficiency, the mutant M145I-214A-229T-247T-317I has a great potential as a detergent additive, and this structure-guided systems engineering strategy may be useful for the protein engineering of the other microbial enzymes to fulfill industrial requirements.     

Interdomain Hydrophobic Interactions Modulate the Thermostability of Microbial Esterases from the Hormone-Sensitive Lipase Family

Microbial hormone-sensitive lipases (HSLs) contain a CAP domain and a catalytic domain. However, it remains unclear how the CAP domain interacts with the catalytic domain to maintain the stability of microbial HSLs. Here, we isolated an HSL esterase, E40, from a marine sedimental metagenomic library. E40 exhibited the maximal activity at 45 °C and was quite thermolabile, with a half-life of only 2 min at 40 °C, which may be an adaptation of E40 to the permanently cold sediment environment. The structure of E40 was solved to study its thermolability. Structural analysis showed that E40 lacks the interdomain hydrophobic interactions between loop 1 of the CAP domain and α7 of the catalytic domain compared with its thermostable homologs. Mutational analysis showed that the introduction of hydrophobic residues Trp²⁰² and Phe²⁰³ in α7 significantly improved E40 stability and that a further introduction of hydrophobic residues in loop 1 made E40 more thermostable because of the formation of interdomain hydrophobic interactions. Altogether, the results indicate that the absence of interdomain hydrophobic interactions between loop 1 and α7 leads to the thermolability of E40. In addition, a comparative analysis of the structures of E40 and other thermolabile and thermostable HSLs suggests that the interdomain hydrophobic interactions between loop 1 and α7 are a key element for the thermostability of microbial HSLs. Therefore, this study not only illustrates the structural element leading to the thermolability of E40 but also reveals a structural determinant for HSL thermostability.     

Structural Elucidation of the Cyclization Mechanism of α-1,6-Glucan by Bacillus circulans T-3040 Cycloisomaltooligosaccharide Glucanotransferase

Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase belongs to the glycoside hydrolase family 66 and catalyzes an intramolecular transglucosylation reaction that produces cycloisomaltooligosaccharides from dextran. The crystal structure of the core fragment from Ser-39 to Met-738 of B. circulans T-3040 cycloisomaltooligosaccharide glucanotransferase, devoid of its N-terminal signal peptide and C-terminal nonconserved regions, was determined. The structural model contained one catalytic (β/α)₈-barrel domain and three β-domains. Domain N with an immunoglobulin-like β-sandwich fold was attached to the N terminus; domain C with a Greek key β-sandwich fold was located at the C terminus, and a carbohydrate-binding module family 35 (CBM35) β-jellyroll domain B was inserted between the 7th β-strand and the 7th α-helix of the catalytic domain A. The structures of the inactive catalytic nucleophile mutant enzyme complexed with isomaltohexaose, isomaltoheptaose, isomaltooctaose, and cycloisomaltooctaose revealed that the ligands bound in the catalytic cleft and the sugar-binding site of CBM35. Of these, isomaltooctaose bound in the catalytic site extended to the second sugar-binding site of CBM35, which acted as subsite −8, representing the enzyme·substrate complex when the enzyme produces cycloisomaltooctaose. The isomaltoheptaose and cycloisomaltooctaose bound in the catalytic cleft with a circular structure around Met-310, representing the enzyme·product complex. These structures collectively indicated that CBM35 functions in determining the size of the product, causing the predominant production of cycloisomaltooctaose by the enzyme. The canonical sugar-binding site of CBM35 bound the mid-part of isomaltooligosaccharides, indicating that the original function involved substrate binding required for efficient catalysis.     

Highly conserved modified nucleosides influence Mg2+-dependent tRNA folding

Transfer RNA structure involves complex folding interactions of the TΨC domain with the D domain. However, the role of the highly conserved nucleoside modifications in the TΨC domain, rT₅₄, Ψ₅₅ and m⁵C₄₉, in tertiary folding is not understood. To determine whether these modified nucleosides have a role in tRNA folding, the association of variously modified yeast tRNA^Phe T-half molecules (nucleosides 40–72) with the corresponding unmodified D-half molecule (nucleosides 1–30) was detected and quantified using a native polyacrylamide gel mobility shift assay. Mg²⁺ was required for formation and maintenance of all complexes. The modified T-half folding interactions with the D-half resulted in K_ds (rT₅₄ = 6 ± 2, m⁵C₄₉ = 11 ± 2, Ψ₅₅ = 14 ± 5, and rT₅₄,Ψ₅₅ = 11 ± 3 µM) significantly lower than that of the unmodified T-half (40 ± 10 µM). However, the global folds of the unmodified and modified complexes were comparable to each other and to that of an unmodified yeast tRNA^Phe and native yeast tRNA^Phe, as determined by lead cleavage patterns at U₁₇ and nucleoside substitutions disrupting the Levitt base pair. Thus, conserved modifications of tRNA’s TΨC domain enhanced the affinity between the two half-molecules without altering the global conformation indicating an enhanced stability to the complex and/or an altered folding pathway.     

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit ε with the catalytic complex of Escherichia coli ATP synthase. Bacterial F-type ATP synthaseis the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit ε could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of ε undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of ε''s conformations is predominant. The assay measures kinetics of ε''s binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound ε to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on ε''s conformational changes.     

F1-ATPase of Escherichia coli: THE ?-INHIBITED STATE FORMS AFTER ATP HYDROLYSIS,IS DISTINCT FROM THE ADP-INHIBITED STATE,AND RESPONDS DYNAMICALLY TO CATALYTIC SITE LIGANDS*

F₁-ATPase is the catalytic complex of rotary nanomotor ATP synthases. Bacterial ATP synthases can be autoinhibited by the C-terminal domain of subunit ϵ, which partially inserts into the enzyme''s central rotor cavity to block functional subunit rotation. Using a kinetic, optical assay of F₁·ϵ binding and dissociation, we show that formation of the extended, inhibitory conformation of ϵ (ϵ_X) initiates after ATP hydrolysis at the catalytic dwell step. Prehydrolysis conditions prevent formation of the ϵ_X state, and post-hydrolysis conditions stabilize it. We also show that ϵ inhibition and ADP inhibition are distinct, competing processes that can follow the catalytic dwell. We show that the N-terminal domain of ϵ is responsible for initial binding to F₁ and provides most of the binding energy. Without the C-terminal domain, partial inhibition by the ϵ N-terminal domain is due to enhanced ADP inhibition. The rapid effects of catalytic site ligands on conformational changes of F₁-bound ϵ suggest dynamic conformational and rotational mobility in F₁ that is paused near the catalytic dwell position.     

In Silico Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

High thermostability is required for alkaline α-amylases to maintain high catalytic activity under the harsh conditions used in textile production. In this study, we attempted to improve the thermostability of an alkaline α-amylase from Alkalimonas amylolytica through in silico rational design and systems engineering of disulfide bridges in the catalytic domain. Specifically, 7 residue pairs (P35-G426, Q107-G167, G116-Q120, A147-W160, G233-V265, A332-G370, and R436-M480) were chosen as engineering targets for disulfide bridge formation, and the respective residues were replaced with cysteines. Three single disulfide bridge mutants—P35C-G426C, G116C-Q120C, and R436C-M480C—of the 7 showed significantly enhanced thermostability. Combinational mutations were subsequently assessed, and the triple mutant P35C-G426C/G116C-Q120C/R436C-M480C showed a 6-fold increase in half-life at 60°C and a 5.2°C increase in melting temperature compared with the wild-type enzyme. Interestingly, other biochemical properties of this mutant also improved: the optimum temperature increased from 50°C to 55°C, the optimum pH shifted from 9.5 to 10.0, the stable pH range extended from 7.0 to 11.0 to 6.0 to 12.0, and the catalytic efficiency (k_cat/K_m) increased from 1.8 × 10⁴ to 2.4 × 10⁴ liters/g · min. The possible mechanism responsible for these improvements was explored through comparative analysis of the model structures of wild-type and mutant enzymes. The disulfide bridge engineering strategy used in this work may be applied to improve the thermostability of other industrial enzymes.     

Complete integrin headpiece opening in eight steps

Carefully soaking crystals with Arg-Gly-Asp (RGD) peptides, we captured eight distinct RGD-bound conformations of the α_IIbβ₃ integrin headpiece. Starting from the closed βI domain conformation, we saw six intermediate βI conformations and finally the fully open βI with the hybrid domain swung out in the crystal lattice. The β1-α1 backbone that hydrogen bonds to the Asp side chain of RGD was the first element to move followed by adjacent to metal ion-dependent adhesion site Ca²⁺, α1 helix, α1’ helix, β6-α7 loop, α7 helix, and hybrid domain. We define in atomic detail how conformational change was transmitted over long distances in integrins, 40 Å from the ligand binding site to the opposite end of the βI domain and 80 Å to the far end of the hybrid domain. During these movements, RGD slid in its binding groove toward α_IIb, and its Arg side chain became ordered. RGD concentration requirements in soaking suggested a >200-fold higher affinity after opening. The thermodynamic cycle shows how higher affinity pays the energetic cost of opening.     

Isolation of Cellulolytic Anaerobic Extreme Thermophiles from New Zealand Thermal Sites

Avicel enrichment cultures from 47 thermal-pool sites in the New Zealand Rotorua-Taupo region were screened for growth and carboxymethyl cellulase activity at 75°C. Eight anaerobic cellulolytic cultures were obtained. The effect of temperature on carboxymethyl cellulase activity was measured, and bacteria were isolated from the five best cultures. Bacteria from two sources designated TP8 and TP10 grew at 75°C, accumulated reducing sugar in the growth medium and gave free cellulases with avicelase activity. Bacteria from sources designated Tok4, Tok8, and Wai21 grew at 75°C, accumulated no free sugars in the medium, and gave free carboxymethyl cellulases with virtually no avicelase activity. All were obligate anaerobic nonsporeforming rods which stained gram negative, grew on pentoses as well as hexoses, and gave ethanol and acetate as major fermentation end products. The isolated strain which produced the most active and stable cellulases (trivially designated TP8.T) had lower rates of free endocellulase accumulation at 75°C than did Clostridium thermocellum at 60°C, but its cellulase activity against avicel and filter paper in culture supernatants was comparable. Tested at 85°C, TP8.T carboxymethyl cellulases included components which were very stable, whereas C. thermocellum carboxymethyl cellulases were all rapidly inactivated. The TP8.T avicelase activity was relatively unaffected by Triton X-100, EDTA, and dithiothreitol. Evidence was obtained for the existence of unisolated, cellulolytic extreme thermophiles producing cellulases which were more stable and active than those from TP8.T.     

Structural and Thermodynamic Characterization of Cadherin·β-Catenin·α-Catenin Complex Formation

The classical cadherin·β-catenin·α-catenin complex mediates homophilic cell-cell adhesion and mechanically couples the actin cytoskeletons of adjacent cells. Although α-catenin binds to β-catenin and to F-actin, β-catenin significantly weakens the affinity of α-catenin for F-actin. Moreover, α-catenin self-associates into homodimers that block β-catenin binding. We investigated quantitatively and structurally αE- and αN-catenin dimer formation, their interaction with β-catenin and the cadherin·β-catenin complex, and the effect of the α-catenin actin-binding domain on β-catenin association. The two α-catenin variants differ in their self-association properties: at physiological temperatures, αE-catenin homodimerizes 10× more weakly than does αN-catenin but is kinetically trapped in its oligomeric state. Both αE- and αN-catenin bind to β-catenin with a K_d of 20 nm, and this affinity is increased by an order of magnitude when cadherin is bound to β-catenin. We describe the crystal structure of a complex representing the full β-catenin·αN-catenin interface. A three-dimensional model of the cadherin·β-catenin·α-catenin complex based on these new structural data suggests mechanisms for the enhanced stability of the ternary complex. The C-terminal actin-binding domain of α-catenin has no influence on the interactions with β-catenin, arguing against models in which β-catenin weakens actin binding by stabilizing inhibitory intramolecular interactions between the actin-binding domain and the rest of α-catenin.     

Structural insights into the substrate specificity of two esterases from the thermophilic Rhizomucor miehei

Two hormone-sensitive lipase (HSL) family esterases (RmEstA and RmEstB) from the thermophilic fungus Rhizomucor miehei, exhibiting distinct substrate specificity, have been recently reported to show great potential in industrial applications. In this study, the crystal structures of RmEstA and RmEstB were determined at 2.15 Å and 2.43 Å resolutions, respectively. The structures of RmEstA and RmEstB showed two distinctive domains, a catalytic domain and a cap domain, with the classical α/β-hydrolase fold. Catalytic triads consisting of residues Ser161, Asp262, and His292 in RmEstA, and Ser164, Asp261, and His291 in RmEstB were found in the respective canonical positions. Structural comparison of RmEstA and RmEstB revealed that their distinct substrate specificity might be attributed to their different substrate-binding pockets. The aromatic amino acids Phe222 and Trp92, located in the center of the substrate-binding pocket of RmEstB, blocked this pocket, thus narrowing its catalytic range for substrates (C2–C8). Two mutants (F222A and W92F in RmEstB) showing higher catalytic activity toward long-chain substrates further confirmed the hypothesized interference. This is the first report of HSL family esterase structures from filamentous fungi.jlr The information on structure-function relationships could open important avenues of exploration for further industrial applications of esterases.     

Heat,Acid and Chemically Induced Unfolding Pathways,Conformational Stability and Structure-Function Relationship in Wheat α-Amylase

Wheat α-amylase, a multi-domain protein with immense industrial applications, belongs to α+β class of proteins with native molecular mass of 32 kDa. In the present study, the pathways leading to denaturation and the relevant unfolded states of this multi-domain, robust enzyme from wheat were discerned under the influence of temperature, pH and chemical denaturants. The structural and functional aspects along with thermodynamic parameters for α-amylase unfolding were probed and analyzed using fluorescence, circular dichroism and enzyme assay methods. The enzyme exhibited remarkable stability up to 70°C with tendency to aggregate at higher temperature. Acid induced unfolding was also incomplete with respect to the structural content of the enzyme. Strong ANS binding at pH 2.0 suggested the existence of a partially unfolded intermediate state. The enzyme was structurally and functionally stable in the pH range 4.0–9.0 with 88% recovery of hydrolytic activity. Careful examination of biophysical properties of intermediate states populated in urea and GdHCl induced denaturation suggests that α-amylase unfolding undergoes irreversible and non-coincidental cooperative transitions, as opposed to previous reports of two-state unfolding. Our investigation highlights several structural features of the enzyme in relation to its catalytic activity. Since, α-amylase has been comprehensively exploited for use in a range of starch-based industries, in addition to its physiological significance in plants and animals, knowledge regarding its stability and folding aspects will promote its biotechnological applications.     

Proofreading exonuclease on a tether: the complex between the E. coli DNA polymerase III subunits α, ε, θ and β reveals a highly flexible arrangement of the proofreading domain

A complex of the three (αεθ) core subunits and the β₂ sliding clamp is responsible for DNA synthesis by Pol III, the Escherichia coli chromosomal DNA replicase. The 1.7 Å crystal structure of a complex between the PHP domain of α (polymerase) and the C-terminal segment of ε (proofreading exonuclease) subunits shows that ε is attached to α at a site far from the polymerase active site. Both α and ε contain clamp-binding motifs (CBMs) that interact simultaneously with β₂ in the polymerization mode of DNA replication by Pol III. Strengthening of both CBMs enables isolation of stable αεθ:β₂ complexes. Nuclear magnetic resonance experiments with reconstituted αεθ:β₂ demonstrate retention of high mobility of a segment of 22 residues in the linker that connects the exonuclease domain of ε with its α-binding segment. In spite of this, small-angle X-ray scattering data show that the isolated complex with strengthened CBMs has a compact, but still flexible, structure. Photo-crosslinking with p-benzoyl-L-phenylalanine incorporated at different sites in the α-PHP domain confirm the conformational variability of the tether. Structural models of the αεθ:β₂ replicase complex with primer-template DNA combine all available structural data.     

X-ray structure of human acid-beta-glucosidase,the defective enzyme in Gaucher disease

Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the gene that encodes acid-β-glucosidase (GlcCerase). Type 1 is characterized by hepatosplenomegaly, and types 2 and 3 by early or chronic onset of severe neurological symptoms. No clear correlation exists between the ~200 GlcCerase mutations and disease severity, although homozygosity for the common mutations N370S and L444P is associated with non- neuronopathic and neuronopathic disease, respectively. We report the X-ray structure of GlcCerase at 2.0 Å resolution. The catalytic domain consists of a (β/α)₈ TIM barrel, as expected for a member of the glucosidase hydrolase A clan. The distance between the catalytic residues E235 and E340 is consistent with a catalytic mechanism of retention. N370 is located on the longest α-helix (helix 7), which has several other mutations of residues that point into the TIM barrel. Helix 7 is at the interface between the TIM barrel and a separate immunoglobulin-like domain on which L444 is located, suggesting an important regulatory or structural role for this non-catalytic domain. The structure provides the possibility of engineering improved GlcCerase for enzyme-replacement therapy, and for designing structure-based drugs aimed at restoring the activity of defective GlcCerase.     

Characterization of an Invertase with pH Tolerance and Truncation of Its N-Terminal to Shift Optimum Activity toward Neutral pH

Most invertases identified to date have optimal activity at acidic pH, and are intolerant to neutral or alkaline environments. Here, an acid invertase named uninv2 is described. Uninv2 contained 586 amino acids, with a 100 amino acids N-terminal domain, a catalytic domain and a C-terminal domain. With sucrose as the substrate, uninv2 activity was optimal at pH 4.5 and at 45°C. Removal of N-terminal domain of uninv2 has shifted the optimum pH to 6.0 while retaining its optimum temperaure at 45°C. Both uninv2 and the truncated enzyme retained highly stable at neutral pH at 37°C, and they were stable at their optimum pH at 4°C for as long as 30 days. These characteristics make them far superior to invertase from Saccharomyces cerevisiae, which is mostly used as industrial enzyme.     

Isomaltulose Synthase from Klebsiella sp. Strain LX3: Gene Cloning and Characterization and Engineering of Thermostability

The gene (palI) encoding isomaltulose synthase (PalI) from a soil bacterial isolate, Klebsiella sp. strain LX3, was cloned and characterized. PalI converts sucrose into isomaltulose, trehalulose, and trace amounts of glucose and fructose. Sequence domain analysis showed that PalI contains an α-amylase domain and (β/α)₈-barrel structures, suggesting that it belongs to the α-amylase family. Sequence alignment indicated that the five amino acid residues of catalytic importance in α-amylases and glucosyltransferases (Asp²⁴¹, Glu²⁹⁵, Asp³⁶⁹, His¹⁴⁵, and His³⁶⁸) are conserved in PalI. Purified recombinant PalI displayed high catalytic efficiency, with a K_m of 54.6 ± 1.7 mM for sucrose, and maximum activity (approximately 328.0 ± 2.5 U/mg) at pH 6.0 and 35°C. PalI activity was strongly inhibited by Fe³⁺ and Hg²⁺ and was enhanced by Mn²⁺ and Mg²⁺. The half-life of PalI was 1.8 min at 50°C. Replacement of selected amino acid residues by proline significantly increased the thermostability of PalI. Simultaneous replacement of Glu⁴⁹⁸ and Arg³¹⁰ with proline resulted in an 11-fold increase in the half-life of PalI at 50°C.     

Structural Analysis of Saccharomyces cerevisiae α-Galactosidase and Its Complexes with Natural Substrates Reveals New Insights into Substrate Specificity of GH27 Glycosidases

α-Galactosidases catalyze the hydrolysis of terminal α-1,6-galactosyl units from galacto-oligosaccharides and polymeric galactomannans. The crystal structures of tetrameric Saccharomyces cerevisiae α-galactosidase and its complexes with the substrates melibiose and raffinose have been determined to 1.95, 2.40, and 2.70 Å resolution. The monomer folds into a catalytic (α/β)₈ barrel and a C-terminal β-sandwich domain with unassigned function. This pattern is conserved with other family 27 glycosidases, but this enzyme presents a unique 45-residue insertion in the β-sandwich domain that folds over the barrel protecting it from the solvent and likely explaining its high stability. The structure of the complexes and the mutational analysis show that oligomerization is a key factor in substrate binding, as the substrates are located in a deep cavity making direct interactions with the adjacent subunit. Furthermore, docking analysis suggests that the supplementary domain could be involved in binding sugar units distal from the scissile bond, therefore ascribing a role in fine-tuning substrate specificity to this domain. It may also have a role in promoting association with the polymeric substrate because of the ordered arrangement that the four domains present in one face of the tetramer. Our analysis extends to other family 27 glycosidases, where some traits regarding specificity and oligomerization can be formulated on the basis of their sequence and the structures available. These results improve our knowledge on the activity of this important family of enzymes and give a deeper insight into the structural features that rule modularity and protein-carbohydrate interactions.     

Role of the NC-Loop in Catalytic Activity and Stability in Lipase from Fervidobacterium changbaicum

Flexible NC-loops between the catalytic domain and the cap domain of the α/β hydrolase fold enzymes show remarkable diversity in length, sequence, and configuration. Recent investigations have suggested that the NC-loop might be involved in catalysis and substrate recognition in many enzymes from the α/β hydrolase fold superfamily. To foster a deep understanding of its role in catalysis, stability, and divergent evolution, we here systemically investigated the function of the NC-loop (residues 131–151) in a lipase (FClip1) from thermophilic bacterium Fervidobacterium changbaicum by loop deletion, alanine-scanning mutagenesis and site-directed mutagenesis. We found that the upper part of the NC-loop (residues 131–138) was of great importance to enzyme catalysis. Single substitutions in this region could fine-tune the activity of FClip1 as much as 41-fold, and any deletions from this region rendered the enzyme completely inactive. The lower part of the NC-loop (residues 139–151) was capable of enduring extensive deletions without loss of activity. The shortened mutants in this region were found to show both improved activity and increased stability simultaneously. We therefore speculated that the NC-loop, especially the lower part, would be a perfect target for enzyme engineering to optimize the enzymatic properties, and might present a hot zone for the divergent evolution of α/β hydrolases. Our findings may provide an opportunity for better understanding of the mechanism of divergent evolution in the α/β hydrolase fold superfamily, and may also guide the design of novel biocatalysts for industrial applications.