Structure Analysis of a New Psychrophilic Marine Protease

Si-Cai Zhang,Xiao-Jian Hu,Qi-Hai Wang,Sheng-Xiang Lin,Yi Han,Tang Li,Mi Sun,Ming Zhou,Jian-Hua Hao,Franca Fraternali

doi:10.1371/journal.pone.0026939

Abstract

A new psychrophilic marine protease was found from a marine bacterium Flavobacterium YS-80 in the Chinese Yellow Sea. The protease is about 49 kD with an isoelectric point about 4.5. It consists of 480 amino acids and is homologous to a psychrophilic alkaline protease (PAP) from an Antarctic Pseudomonas species. The protein was purified from the natural bacterium fermented and crystallized. Its crystal structure (PDB ID 3U1R) was solved at 2.0 Å by Molecular Replacement using a model based on PAP, and was refined to a crystallographic Rwork of 0.16 and an Rfree of 0.21. The marine protease consists of a two domain structure with an N-terminal domain including residues 37–264 and a C-terminal domain including residues 265–480. Similar to PAP, the N-terminal domain is responsible for proteolysis and the C-terminal is for stability. His186, His190, His196 and Tyr226 are ligands for the Zn2+ ion in the catalytic center. The enzyme's Tyr226 is closer to the Zn2+ ion than in PAP and it shows a stronger Zn2+―Tyr-OH bond. There are eight calcium ions in the marine protease molecule and they have significantly shorter bond distances to their ligands compared to their counterparts in all three crystal forms of PAP. On the other hand, the loops in the marine protease are more compact than in PAP. This makes the total structure stable and less flexible, resulting in higher thermo stability. These properties are consistent with the respective environments of the proteases. The structural analysis of this new marine protease provides new information for the study of psychrophilic proteases and is helpful for elucidating the structure-environment adaptation of these enzymes.

Highlights

Many extreme microbes have been found on the seabed
marine protease (MP) consists of 480 amino acids, is homologous to psychrophilic alkaline protease (PAP, from an Antarctic Pseudomonas species), a mesophilic counterpart alkaline protease (AP, from P. aeruginosa) and serralysin (SMP, from S. marcescens) according to sequence alignment (Fig. 1)
Overall structure The MP crystal structure (PDB ID 3U1R) with 456 amino acid residues was solved by molecular replacement using the structure of PAP form 3 (PDB ID 1OMJ) as a model, and refined to 2.0 Aresolution with an Rwork of 0.16 and an Rfree of 0.21

Summary

Introduction

Many extreme microbes have been found on the seabed. These organisms live at temperatures close to freezing point. Several alkaline proteases have been found in different locations and have been crystallized, including the psychrophilic enzymes and their mesophilic counterparts. Structural studies of these proteases are important to elucidate the structure-environment adaptation of proteins. It belongs to the serralysin family comprising a group of bacterial metalloproteases.[6,7,8,9] The MP displays 88% sequence identity with PAP, and 60% with AP This protease is a secreted protein that has a molecular mass of 49320 Daltons and an isoelectric point of about 4.5. The crystal structure of unliganded AP (PDB ID 1AKL) from P. aeruginosa was determined at 2.0 Aby X-ray method.[7]

Methods

Results

Conclusion