Evidence for the participation of an extra α-helix at β-subunit surface in the thermal stability of Co-type nitrile hydratase

Abstract

Nitrile hydratase (NHase) has attracted considerable attention since it can efficiently catalyze the hydration of nitriles to valuable amides. However, the poor stability of NHase is one of the main drawbacks in the industrial application. In this study, we compared the structural difference between Fe-type and Co-type NHase and found that an extra α helix existed at the β-subunit surface of Co-type NHase (defined as the β-6th helix). Then, the effects of the β-6th helix were investigated on the thermal stability and the catalytic kinetics of a Co-type NHase from Aurantimonas manganoxydans ATCC BAA-1229 (NHase1229). When the β-6th helix was deleted or disrupted, the thermal stability of NHase1229 was reduced to 17.6 and 12.9% of that of wild NHase1229, respectively. Thus, the β-6th helix is important for the thermal stability of Co-type NHase. Based on the structural characteristics of Co-type NHase, the β-6th helix may be interacted with another helix at the α-subunit (defined as the α-2nd helix) by hydrophobic network just as a "magnetic suction buckle" on the enzyme surface to stabilize the binding of α- and β-subunits. The β-6th helix is located at the mouth of the substrate and product tunnel, so it plays crucial roles in catalytic process. Furthermore, the β-6th helix in NHase1229 was swapped with a thermophilic NHase fragment from Pseudonocardia thermophila JCM3095 (NHase1229-Swap). The thermal stability of NHase1229-Swap was significantly improved, and the half-life was approximately 2.4-fold at 40 °C than that of the wild NHase1229. The knowledge is useful for improving the stability of NHases by restriction fragment swapping.

Computational Design of Nitrile Hydratase from Pseudonocardia thermophila JCM3095 for Improved Thermostability

“…This was the best reported result so far for thermostability improvement of un-immobilized and natural NHase. Pei in his report enhanced the thermostability of NHase from Aurantimonas manganoxydans ATCC BAA-1229 from 60 min up to 100 min, measured at 50 °C (like in the present study) [ 9 ].…”

“…In this study, we showed that applying an online tool, that is easy to use for all proteins and which proposes introducing several point mutations, is helpful in designing enzymes with improved thermostability. Our newly designed Pt NHase showed even better thermostability improvement than those reported for natural NHase by Pei [ 9 ].…”

“…The modulated enzyme showed improved thermostability. The modified NHase exhibited 50% of activity after 100 min at 50 °C (under the same condition, wild-type (WT) showed 50% of activity after 60 min) [ 9 ]. However, exchanging protein fragments, as applied by Pei, is very challenging and not possible in all cases.…”

Structure guided protein engineering increases enzymatic activities of the SGNH family esterases

“…S5). According to previous studies, the thermal stability can be affected by (i) more abundant, high hydrophobicity, charged residues (such as Glu, Arg, and Lys) rather than uncharged polar amino acid (such as Ser, Thr, Asn, and Gln) in the amino acid sequences [41,48,49]; (ii) more α-helices in structures [50,51]; and (iii) some intermolecular forces, such as hydrogen bonds [52]. AlinE4 has six glutamine residues and twelve lysine residues, while CrmE10 has twelve glutamine residues and only one lysine residue, which might lead to the difference in thermal stability.…”

Structure guided protein engineering increases enzymatic activities of the SGNH family esterases

“…S5). According to previous studies, the thermal stability can be affected by (i) more abundant, high hydrophobicity, charged residues (such as Glu, Arg, and Lys) rather than uncharged polar amino acid (such as Ser, Thr, Asn, and Gln) in the amino acid sequences [40, 47,48]; (ii) more α-helices in structures [49,50];…”