Engineering a Disulfide Bond in the Lid Hinge Region of <em>Rhizopus chinensis</em> Lipase: Increased Thermostability and Altered Acyl Chain Length Specificity

<div><p>The key to enzyme function is the maintenance of an appropriate balance between molecular stability and structural flexibility. The lid domain which is very important for “interfacial activation” is the most flexible part in the lipase structure. In this work, rational design was applied to explore the relationship between lid rigidity and lipase activity by introducing a disulfide bond in the hinge region of the lid, in the hope of improving the thermostability of <em>R. chinensis</em> lipase through stabilization of the lid domain without interfering with its catalytic performance. A disulfide bridge between F95C and F214C was introduced into the lipase from <em>R. chinensis</em> in the hinge region of the lid according to the prediction of the “Disulfide by Design” algorithm. The disulfide variant showed substantially improved thermostability with an eleven-fold increase in the <em>t</em>_1/2 value at 60°C and a 7°C increase of <em>T</em>_m compared with the parent enzyme, probably contributed by the stabilization of the geometric structure of the lid region. The additional disulfide bond did not interfere with the catalytic rate (<em>k</em>_cat) and the catalytic efficiency towards the short-chain fatty acid substrate, however, the catalytic efficiency of the disulfide variant towards pNPP decreased by 1.5-fold probably due to the block of the hydrophobic substrate channel by the disulfide bond. Furthermore, in the synthesis of fatty acid methyl esters, the maximum conversion rate by RCLCYS reached 95% which was 9% higher than that by RCL. This is the first report on improving the thermostability of the lipase from <em>R. chinensis</em> by introduction of a disulfide bond in the lid hinge region without compromising the catalytic rate.</p> </div

Enzyme properties of RCLCYS and RCL.

<p>Enzyme properties of RCLCYS and RCL.</p

EMSA of lamprey HMGB1 binding to double-stranded polynucleotides.

<p>Double-stranded lamprey GAPDH DNA (100 ng, 101 bp and 12 bp in length) was incubated with purified Lj-HMGB1 protein at various concentrations, and aliquots were taken for electrophoresis on a 2% agarose gel (A) and a 20% native PAGE gel (B). Lane 1, Tris buffer; Lane 2, 10 ng of Lj-HMGB1; Lane 3, 20 ng of Lj-HMGB1; Lane 4, 30 ng of Lj-HMGB1; Lane 5, 30 ng of BSA; Lane 6, 10 ng of human HMGB1. (C). DNA hydrolysis in the presence of rLj-HMGB1. pEGFP-N1 DNA (4730 bp, 100 ng) was hydrolyzed by DNase I in the presence of rLj-HMGB1. Tris buffer (lane 2), BSA (lane 4), rLj-HMGB1 (lane 6) or human HMGB1 (lane 8) were incubated with pEGFP-N1 DNA at a quantitative ratio of 1∶10 in 20 mM Tris-HCl buffer containing 2 mM MgCl₂ (pH 8.0) at room temperature for 10 min. DNase I (0.05 units, TaKaRa) was then added to each sample to hydrolyze pEGFP-N1 DNA at 37 °C for 10 min. Tris buffer (lane 1), BSA (lane 3), rLj-HMGB1 (lane 5) or human HMGB1 (lane 7) incubated with pEGFP-N1 DNA without DNase I served as the controls. The aliquots were analyzed on 1% agarose gels.</p

Phylogenetic relationship of lamprey HMGB1 with other HMGB members.

<p>A phylogenetic tree was constructed based on the amino acid sequences of HMGB from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035755#pone-0035755-g002" target="_blank">Fig. 2</a>. The number at each node indicates the percentage of bootstrapping after 1000 replications. The bar (0.05) indicates genetic distance.</p

Primers for plasmid pPIC9K-proRCLCYS construction.

<p>Primers for plasmid pPIC9K-proRCLCYS construction.</p

Heat-induced denaturation of RCLCYS and RCL.

<p>CD spectra of RCLCYS (•) and RCL (▪) were recorded at 220 nm at the temperatures indicated.</p

Time course of solvent-free conversion of soy bean oil to fatty acid methyl esters by RCLCYS and RCL at 40°C.

<p>Time course of solvent-free conversion of soy bean oil to fatty acid methyl esters by RCLCYS and RCL at 40°C.</p

Thermal inactivation of RCLCYS and RCL at 60°C.

<p>Thermal inactivation of RCLCYS and RCL at 60°C.</p

Models of the simulated structure of RCL in a close form.

<p>Red, the lid hinge regions (⁸²GTNS⁸⁵ and ⁹²DMVFT⁹⁶), F214/F95 and N84/G266 as ball and stick; Blue, the catalytic triad of S145-H257-D204; Green, three original disulfide bonds; Pink, the free cysteine C177; Purple, the lid region (⁸⁶FRSAIT⁹¹).</p

Sequence alignment of Lj-HMGB1 with HMGB1/2/3 of other species using ClustalX.

<p>The accession numbers of the amino acid sequences extracted from the EXPASY database are as follows: human HMGB1 (P09429); mouse HMGB1 (P63158); chick HMGB1 (Q9PUK9); frog HMGB1 (Q7SZ42); human HMGB2 (P26583); mouse HMGB2 (P30681); chick HMGB2 (P26584); frog HMGB2 (Q32NS7); human HMGB3 (O15347); mouse HMGB3 (O54879); chick HMGB3 (P40618); frog HMGB3 (Q1XCD9); Lj-HMGB1 (<i>Lampetra japonica</i>, HQ615991); Lf-HMGB1 (<i>Lampetra fluviatilis</i>, Q91070); amphioxus HMG1/2 (Q6PUE4); and sea urchin HMG1 (P40644). Identical (<i>asterisk</i>) and similar (<i>colon</i>) residues are indicated. <i>Dashes</i> represent gaps inserted into the alignment.</p