Dataset Information

Roles of Active-Site Aromatic Residues in Cold Adaptation of Sphingomonas glacialis Esterase EstSP1.

ABSTRACT: The aromatic amino acids, Tyr or Trp, which line the active-site walls of esterases, stabilize the catalytic His loop via hydrogen bonding. A Tyr residue is preferred in extremophilic esterases (psychrophilic or hyperthermophilic esterases), whereas a Trp residue is preferred in moderate-temperature esterases. Here, we provide evidence that Tyr and Trp play distinct roles in cold adaptation of the psychrophilic esterase EstSP1 isolated from an Arctic bacterium Sphingomonas glacialis PAMC 26605. Stern-Volmer plots showed that the mutation of Tyr191 to Ala, Phe, Trp, and His resulted in reduced conformational flexibility of the overall protein structure. Interestingly, the Y191W and Y191H mutants showed increased thermal stability at moderate temperatures. All Tyr191 mutants showed reduced catalytic activity relative to wild-type EstSP1. Our results indicate that Tyr with its phenyl hydroxyl group is favored for increased conformational flexibility and high catalytic activity of EstSP1 at low temperatures at the expense of thermal stability. The results of this study suggest that, in the permanently cold Arctic zone, enzyme activity has been selected for psychrophilic enzymes over thermal stability. The results presented herein provide novel insight into the roles of Tyr and Trp residues for temperature adaptation of enzymes that function at low, moderate, and high temperatures.

SUBMITTER: Kashif A

PROVIDER: S-EPMC6645578 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Roles of Active-Site Aromatic Residues in Cold Adaptation of <i>Sphingomonas glacialis</i> Esterase EstSP1.

Kashif Ayesha A Tran Lan-Huong LH Jang Sei-Heon SH Lee ChangWoo C

ACS omega 20171208 12

The aromatic amino acids, Tyr or Trp, which line the active-site walls of esterases, stabilize the catalytic His loop via hydrogen bonding. A Tyr residue is preferred in extremophilic esterases (psychrophilic or hyperthermophilic esterases), whereas a Trp residue is preferred in moderate-temperature esterases. Here, we provide evidence that Tyr and Trp play distinct roles in cold adaptation of the psychrophilic esterase EstSP1 isolated from an Arctic bacterium <i>Sphingomonas glacialis</i> PAMC ...[more]

PMID: 31457406

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Project description:Escherichia coli cysteine desulfurase (CD), IscS, modifies basal metabolism by transferring sulphur (S) from L-cysteine to numerous cellular pathways, whereas NFS1, a human CD, is active only in the formation of the [Acp]2:[ISD11]2:[NFS1]2 complex. Despite the accumulation of red-coloured IscS in E. coli cells as a result of the deficiency of accessible iron, as revealed in our previous studies, the mechanism of the potential enzymatic reaction remains unclear. In this study, the N-terminus of IscS was fused with the C-terminus of NFS1, which was reported to be almost fully active as IscS and exhibits a pyridoxal 5'-phosphate (PLP) absorption peak at 395 nm. Moreover, SUMO-EH-IscS exhibited significant growth recovery and NADH-dehydrogenase I activity in the iscS mutant cells. Furthermore, through in vitro and in vivo experiments combined with high-performance liquid chromatography and ultra-performance liquid chromatography-tandem mass spectrometry, it was shown that the new absorption peaks of the IscS H104Q, IscS Q183E, IscS K206A, and IscS K206A&C328S variants at 340 and 350 nm may correspond to the enzyme reaction intermediates, Cys-ketimine and Cys-aldimine, respectively. However, after mutation of the conserved active-site residues, additional absorption peaks at 420 and 430 nm were associated with PLP migration in the active-site pocket. Additionally, the corresponding absorption peaks of Cys-quinonoid, Ala-ketimine, and Ala-aldimine intermediates in IscS were 510, 325, and 345 nm, respectively, as determined by site-directed mutagenesis and substrate/product-binding analyses during the CD reaction process. Notably, red IscS formed in vitro by incubating IscS variants (Q183E and K206A) with excess L-alanine and sulphide under aerobic conditions produced an absorption peak similar to the wild-type IscS, at 510 nm. Interestingly, site-directed mutation of IscS with hydrogen bonds to PLP at Asp180 and Gln183 resulted in a loss of enzymatic activity followed by an absorption peak consistent with NFS1 (420 nm). Furthermore, mutations at Asp180 or Lys206 inhibited the reaction of IscS in vitro with L-cysteine (substrate) and L-alanine (product). These results suggest that the conserved active site residues (His104, Asp180, and Gln183) and their hydrogen bond with PLP in the N-terminus of IscS play a key role in determining whether the L-cysteine substrate can enter the active-site pocket and regulate the enzymatic reaction process. Therefore, our findings provide a framework for evaluating the roles of conserved active-site residues, motifs, and domains in CDs.

Project description:The effects of mutation of key active-site residues (Arg-47, Tyr-51, Phe-42 and Phe-87) in Bacillus megaterium flavocytochrome P450 BM3 were investigated. Kinetic studies on the oxidation of laurate and arachidonate showed that the side chain of Arg-47 contributes more significantly to stabilization of the fatty acid carboxylate than does that of Tyr-51 (kinetic parameters for oxidation of laurate: R47A mutant, Km 859 microM, kcat 3960 min-1; Y51F mutant, Km 432 microM, kcat 6140 min-1; wild-type, Km 288 microM, kcat 5140 min-1). A slightly increased kcat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (DeltaG) resulting from a smaller DeltaG of substrate binding. The side chain of Phe-42 acts as a phenyl 'cap' over the mouth of the substrate-binding channel. With mutant F42A, Km is massively increased and kcat is decreased for oxidation of both laurate (Km 2. 08 mM, kcat 2450 min-1) and arachidonate (Km 34.9 microM, kcat 14620 min-1; compared with values of 4.7 microM and 17100 min-1 respectively for wild-type). Amino acid Phe-87 is critical for efficient catalysis. Mutants F87G and F87Y not only exhibit increased Km and decreased kcat values for fatty acid oxidation, but also undergo an irreversible conversion process from a 'fast' to a 'slow' rate of substrate turnover [for F87G (F87Y)-catalysed laurate oxidation: kcat 'fast', 760 (1620) min-1; kcat 'slow', 48.0 (44.6) min-1; kconv (rate of conversion from fast to slow form), 4.9 (23.8) min-1]. All mutants showed less than 10% uncoupling of NADPH oxidation from fatty acid oxidation. The rate of FMN-to-haem electron transfer was shown to become rate-limiting in all mutants analysed. For wild-type P450 BM3, the rate of FMN-to-haem electron transfer (8340 min-1) is twice the steady-state rate of oxidation (4100 min-1), indicating that other steps contribute to rate limitation. Active-site structures of the mutants were probed with the inhibitors 12-(imidazolyl)dodecanoic acid and 1-phenylimidazole. Mutant F87G binds 1-phenylimidazole >10-fold more tightly than does the wild-type, whereas mutant Y51F binds the haem-co-ordinating fatty acid analogue 12-(imidazolyl)dodecanoic acid >30-fold more tightly than wild-type.

Dataset Information

Roles of Active-Site Aromatic Residues in Cold Adaptation of Sphingomonas glacialis Esterase EstSP1.

Publications

Roles of Active-Site Aromatic Residues in Cold Adaptation of <i>Sphingomonas glacialis</i> Esterase EstSP1.

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