The roles of histidine and tyrosine residues in the active site of collagenase in Grimontia hollisae

Abstract Collagenase from the Grimontia hollisae strain 1706B (Ghcol) is a zinc metalloproteinase with the zinc-binding motif H492EXXH496. It exhibits higher collagen-degrading activity than the collagenase from Clostridium histolyticum, which is widely used in industry. We previously examined the pH and temperature dependencies of Ghcol activity; Glu493 was thought to contribute acidic pKa (pKe1), while no residue was assigned to contribute alkaline pKa (pKe2). In this study, we introduced nine single mutations at the His or Tyr residues in and near the active site. Our results showed that H412A, H485A, Y497A, H578A and H737A retained the activities to hydrolyze collagen and gelatin, while H426A, H492A, H496A and Y568A lacked them. Purification of active variants H412A, H485A, H578A and H737A, along with inactive variants H492A and H496A, were successful. H412A preferred (7-methoxycoumarin-4-yl)acetyl-L-Lys-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2 to collagen, while H485A preferred collagen to the peptide, suggesting that His412 and His485 are important for substrate specificity. Purification of the active variant Y497A and inactive variants H426A and Y568A were unsuccessful, suggesting that these three residues were important for stability. Based on the reported crystal structure of clostridial collagenase, Tyr568 of Ghcol is suggested to be involved in catalysis and may be the ionizable residue for pKe2.

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Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classification as a metzincin

Biochemical Journal ◽

10.1042/bj3580359 ◽

2001 ◽

Vol 358 (2) ◽

pp. 359-367 ◽

Cited By ~ 70

Author(s):

Henning B. BOLDT ◽

Michael T. OVERGAARD ◽

Lisbeth S. LAURSEN ◽

Kathrin WEYER ◽

Lars SOTTRUP-JENSEN ◽

...

Keyword(s):

Plasma Protein ◽

Active Site ◽

Mammalian Cells ◽

Mutational Analysis ◽

Protein A ◽

Residual Activity ◽

Zinc Binding ◽

Binding Motif ◽

Dependent Manner ◽

Zinc Binding Motif

The bioavailability of insulin-like growth factor (IGF)-I and -II is controlled by six IGF-binding proteins (IGFBPs 1–6). Bound IGF is not active, but proteolytic cleavage of the binding protein causes release of IGF. Pregnancy-associated plasma protein-A (PAPP-A) has recently been found to cleave IGFBP-4 in an IGF-dependent manner. To experimentally support the hypothesis that PAPP-A belongs to the metzincin superfamily of metalloproteinases, all containing the elongated zinc-binding motif HEXXHXXGXXH (His-482–His-492 in PAPP-A), we expressed mutants of PAPP-A in mammalian cells. Substitution of Glu-483 with Ala causes a complete loss of activity, defining this motif as part of the active site of PAPP-A. Interestingly, a mutant with Glu-483 replaced by Gln shows residual activity. Known metzincin structures contain a so-called Met-turn, whose strictly conserved Met residue is thought to interact directly with residues of the active site. By further mutagenesis we provide experimental evidence that Met-556 of PAPP-A, 63 residues from the zinc-binding motif, is located in a Met-turn of PAPP-A. Our hypothesis is also supported by secondary-structure prediction, and the ability of a 55-residue deletion mutant (d[S498-Y552]) to express and retain antigenecity. However, because PAPP-A differs in the features defining the individual established metzincin families, we suggest that PAPP-A belongs to a separate family. We also found that PAPP-A can undergo autocleavage, and that autocleaved PAPP-A is inactive. A lack of unifying elements in the sequences around the found cleavage sites of PAPP-A and a variant suggests steric regulation of substrate specificity.

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Crystal Structure of Butyrate Kinase 2 from Thermotoga maritima, a Member of the ASKHA Superfamily of Phosphotransferases

Journal of Bacteriology ◽

10.1128/jb.00906-08 ◽

2009 ◽

Vol 191 (8) ◽

pp. 2521-2529 ◽

Cited By ~ 5

Author(s):

Jiasheng Diao ◽

Miriam S. Hasson

Keyword(s):

Crystal Structure ◽

Substrate Specificity ◽

Active Site ◽

Thermotoga Maritima ◽

Structural Basis ◽

Acetate Kinase ◽

Phosphoryl Transfer ◽

Atp Binding ◽

Binding Motif ◽

Butyrate Kinase

ABSTRACT The enzymatic transfer of phosphoryl groups is central to the control of many cellular processes. One of the phosphoryl transfer mechanisms, that of acetate kinase, is not completely understood. Besides better understanding of the mechanism of acetate kinase, knowledge of the structure of butyrate kinase 2 (Buk2) will aid in the interpretation of active-site structure and provide information on the structural basis of substrate specificity. The gene buk2 from Thermotoga maritima encodes a member of the ASKHA (acetate and sugar kinases/heat shock cognate/actin) superfamily of phosphotransferases. The encoded protein Buk2 catalyzes the phosphorylation of butyrate and isobutyrate. We have determined the 2.5-Å crystal structure of Buk2 complexed with (β,γ-methylene) adenosine 5′-triphosphate. Buk2 folds like an open-shelled clam, with each of the two domains representing one of the two shells. In the open active-site cleft between the N- and C-terminal domains, the active-site residues consist of two histidines, two arginines, and a cluster of hydrophobic residues. The ATP binding region of Buk2 in the C-terminal domain consists of abundant glycines for nucleotide binding, and the ATP binding motif is similar to those of other members of the ASKHA superfamily. The enzyme exists as an octamer, in which four disulfide bonds form between intermolecular cysteines. Sequence alignment and structure superposition identify the simplicity of the monomeric Buk2 structure, a probable substrate binding site, the key residues in catalyzing phosphoryl transfer, and the substrate specificity differences among Buk2, acetate, and propionate kinases. The possible enzyme mechanisms are discussed.

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Zn2+Ions Selectively Induce Antimicrobial Salivary Peptide Histatin-5 To Fuse Negatively Charged Vesicles. Identification and Characterization of a Zinc-Binding Motif Present in the Functional Domain†

Biochemistry ◽

10.1021/bi990212c ◽

1999 ◽

Vol 38 (30) ◽

pp. 9626-9633 ◽

Cited By ~ 51

Author(s):

Sonia Melino ◽

Stefano Rufini ◽

Marco Sette ◽

Roberto Morero ◽

Alessandro Grottesi ◽

...

Keyword(s):

Zinc Binding ◽

Binding Motif ◽

Functional Domain ◽

Histatin 5 ◽

Salivary Peptide ◽

Identification And Characterization ◽

Zinc Binding Motif

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Effect of mutations in a zinc-binding domain of yeast RNA polymerase C (III) on enzyme function and subunit association

Molecular and Cellular Biology ◽

10.1128/mcb.12.3.1087-1095.1992 ◽

1992 ◽

Vol 12 (3) ◽

pp. 1087-1095

Author(s):

M Werner ◽

S Hermann-Le Denmat ◽

I Treich ◽

A Sentenac ◽

P Thuriaux

Keyword(s):

Rna Polymerase ◽

Zinc Binding ◽

Enzyme Function ◽

Directed Mutagenesis ◽

Binding Motif ◽

Zinc Ions ◽

Amino Terminal ◽

Zinc Binding Domain ◽

Zinc Binding Motif

The conserved amino-terminal region of the largest subunit of yeast RNA polymerase C is capable of binding zinc ions in vitro. By oligonucleotide-directed mutagenesis, we show that the putative zinc-binding motif CX2CX6-12CXGHXGX24-37CX2C, present in the largest subunit of all eukaryotic and archaebacterial RNA polymerases, is essential for the function of RNA polymerase C. All mutations in the invariant cysteine and histidine residues conferred a lethal phenotype. We also obtained two conditional thermosensitive mutants affecting this region. One of these produced a form of RNA polymerase C which was thermosensitive and unstable in vitro. This instability was correlated with the loss of three of the subunits which are specific to RNA polymerase C: C82, C34, and C31.

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Structural insights into dihydroxylation of terephthalate, a product of polyethylene terephthalate degradation

Journal of Bacteriology ◽

10.1128/jb.00543-21 ◽

2022 ◽

Author(s):

Jai Krishna Mahto ◽

Neetu Neetu ◽

Monica Sharma ◽

Monika Dubey ◽

Bhanu Prakash Vellanki ◽

...

Keyword(s):

Crystal Structure ◽

Substrate Specificity ◽

Polyethylene Terephthalate ◽

Active Site ◽

Catalytic Domain ◽

Structural Features ◽

Comamonas Testosteroni ◽

Plastic Pollution ◽

Microbial Utilization ◽

Steady State Kinetics

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this Polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis -diol is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO KF1 ). The TPDO KF1 exhibited the substrate specificity for TPA ( k cat / K m = 57 ± 9 mM −1 s −1 ). The TPDO KF1 structure harbors characteristics RO features as well as a unique catalytic domain that rationalizes the enzyme’s function. The docking and mutagenesis studies reveal that its substrate specificity to TPA is mediated by Arg309 and Arg390 residues, two residues positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, its substitution to alanine decreases the activity ( k cat ) by 80%. Together, this study delineates the structural features that dictate the substrate recognition and specificity of TPDO. Importance The global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential in tackling this. Microbial utilization of this released product, TPA is an emerging and promising strategy for waste-to-value creation. Research from the last decade has discovered terephthalate dioxygenase (TPDO), as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we have determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

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