The Thioredoxin Binding Site of Phosphoribulokinase Overlaps the Catalytic Site

Microbial α-glucans produced by GH70 (glycoside hydrolase family 70) glucansucrases are gaining importance because of the mild conditions for their synthesis from sucrose, their biodegradability, and their current and anticipated applications that largely depend on their molar mass. Focusing on the alternansucrase (ASR) from Leuconostoc citreum NRRL B-1355, a well-known glucansucrase catalyzing the synthesis of both high- and low-molar-mass alternans, we searched for structural traits in ASR that could be involved in the control of alternan elongation. The resolution of five crystal structures of a truncated ASR version (ASRΔ2) in complex with different gluco-oligosaccharides pinpointed key residues in binding sites located in the A and V domains of ASR. Biochemical characterization of three single mutants and three double mutants targeting the sugar-binding pockets identified in domain V revealed an involvement of this domain in alternan binding and elongation. More strikingly, we found an oligosaccharide-binding site at the surface of domain A, distant from the catalytic site and not previously identified in other glucansucrases. We named this site surface-binding site (SBS) A1. Among the residues lining the SBS-A1 site, two (Gln700 and Tyr717) promoted alternan elongation. Their substitution to alanine decreased high-molar-mass alternan yield by a third, without significantly impacting enzyme stability or specificity. We propose that the SBS-A1 site is unique to alternansucrase and appears to be designed to bind alternating structures, acting as a mediator between the catalytic site and the sugar-binding pockets of domain V and contributing to a processive elongation of alternan chains.

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Multiple molecular recognition and catalysis. A multifunctional anion receptor bearing an anion binding site, an intercalating group, and a catalytic site for nucleotide binding and hydrolysis

Journal of the American Chemical Society ◽

10.1021/ja00166a025 ◽

1990 ◽

Vol 112 (10) ◽

pp. 3896-3904 ◽

Cited By ~ 177

Author(s):

Mir Wais Hosseini ◽

A. John Blacker ◽

Jean Marie Lehn

Keyword(s):

Molecular Recognition ◽

Binding Site ◽

Nucleotide Binding ◽

Catalytic Site ◽

Anion Binding ◽

Anion Receptor

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Structure ofHaemophilus influenzaeHslV protein at 1.9 Å resolution, revealing a cation-binding site near the catalytic site

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s090744490101575x ◽

2001 ◽

Vol 57 (12) ◽

pp. 1950-1954 ◽

Cited By ~ 16

Author(s):

Marcelo C. Sousa ◽

David B. McKay

Keyword(s):

Binding Site ◽

Catalytic Site ◽

Cation Binding ◽

Cation Binding Site

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Thioredoxin binding site of phosphoribulokinase overlaps the catalytic site. [R]

10.2172/5463659 ◽

1986 ◽

Author(s):

M.A. Porter ◽

F.C. Hartman

Keyword(s):

Binding Site ◽

Catalytic Site

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Organophosphate hydrolase interacts with ferric-enterobactin and promotes iron uptake in association with TonB dependent transport system.

Biochemical Journal ◽

10.1042/bcj20200299 ◽

2020 ◽

Author(s):

Hari Parapatla ◽

Ramurthy Gudla ◽

Guruprasad Varma Konduru ◽

Elsin Raju Devadasu ◽

Hampapathula Adimurthy Nagarajaram ◽

...

Keyword(s):

Binding Site ◽

Active Site ◽

Iron Uptake ◽

Docking Studies ◽

Protein Docking ◽

Catalytic Site ◽

Organophosphate Hydrolase ◽

E Coli ◽

Target Membrane ◽

Dependent Transport

Our previous studies have shown the existence of organophosphate hydrolase (OPH) as a part of the inner membrane associated TonB complex (ExbB/ExbD and TonB) of Sphingobium fuliginis. We now show its involvement in iron uptake by establishing direct interactions with ferric-enterobactin. The interactions between OPH and ferric-enterobactin were not affected even when the active site architecture is altered by substituting active site aspartate with either alanine or asparagine. Protein docking studies further substantiated these findings and predicted the existence of ferric-enterobactin binding site that is different from the catalytic site of OPH. A lysine residue (82 K) found at the predicted ferric-enterobactin binding site facilitated interactions between OPH and ferric-enterobactin. Substitution of lysine with alanine did not affect triesterase activity, but it abrogated OPH ability to interact with both ferric-enterobactin and ExbD, strengthening further the fact that the catalytic site is not the site for binding of these ligands. In the absence of interactions between OPHK82A and ExbD, OPHK82A failed to target membrane in E. coli cells. The Sphingobium fuliginis TonB dependent transport (SfTonBDT) system was reconstituted in E. coli GS027 cells generated by deleting the exbD and tonB genes. The E. coli GS030 cells having SfTonBDT system with OPH showed increased iron uptake. Such an increase was not seen in E. coli GS029, cells having SfTonBDT system generated either by omitting OPH or by including its variants, OPHD301A, OPHD301N suggesting a role for OPH in enhanced iron uptake.

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A re-appraisal of the structural basis of stereochemical recognition in papain. Insensitivity of binding-site-catalytic-site signalling to P2-chirality in a time-dependent inhibition

Biochemical Journal ◽

10.1042/bj2660645 ◽

1990 ◽

Vol 266 (3) ◽

pp. 645-651 ◽

Cited By ~ 6

Author(s):

W Templeton ◽

D Kowlessur ◽

E W Thomas ◽

C M Topham ◽

K Brocklehurst

Keyword(s):

Transition State ◽

Binding Site ◽

Kinetic Data ◽

Model Building ◽

Ph Dependence ◽

Thiol Group ◽

Order Rate Constant ◽

Catalytic Site ◽

Structural Basis ◽

Compound I

1. 2-(N'-Acetyl-D-phenylalanylamino)ethyl 2′-pyridyl disulphide (compound I) [m.p. 123-124 degrees C; [alpha]20D -7.1 degrees (c 0.042 in methanol)] was synthesized, and the results of a study of the pH-dependence of the second-order rate constant (k) for its reaction with the catalytic-site thiol group of papain (EC 3.4.22.2), together with existing kinetic data for the analogous reaction of the L-enantiomer (compound II), were used to evaluate the consequences for transition-state geometry of the difference in chirality at the P2 position of the probe molecule. 2. The kinetic data suggest that the D-enantiomer binds approx. 40-fold less tightly to papain than the L-enantiomer but that the binding-site-catalytic-site signalling that results in a (His-159)-Im(+)-H-assisted transition state occurs equally effectively in the interaction of the former probe as in that of the latter. This results in pH-k profiles for the reactions of both enantiomers each characterized by four macroscopic pKa values (3.7-3.9, 4.1-4.3, 7.9-8.3 and 9.4-9.5) in which k is maximal at pH approx. 6 where the -Im(+)-H-assisted transition state is most fully developed. 3. Model building indicates that both enantiomers can bind to papain such that the phenyl ring of the N-acetylphenylalanyl group makes hydrophobic contacts in the binding pocket of the S2 subsite with preservation of the three hydrogen-bonding interactions involving the substrate analogue reagent and (Asp-158) C = O, (Gly-66) C = O, and (Gly-66)-N-H of papain. Earlier predictions that binding of N-acyl-D-phenylalanine derivatives to papain would be prevented on steric grounds [Berger & Schechter (1970) Philos. Trans. R. Soc. London B 257, 249-264; Lowe & Yuthavong (1971) Biochem. J. 124, 107-115; Lowe (1976) Tetrahedron 32, 291-302] were based on assumed models that are not consistent with the X-ray-diffraction data for papain inhibited by alkylation of Cys-25 with N-benzyloxycarbonyl-Phe-Ala-chloromethane [Drenth, Kalk & Swen (1976) Biochemistry 15, 3731-3738]. 4. The possibility that the kinetic expression of P2-S2 stereospecificity may depend on the nature of the chemistry occurring in the catalytic site of papain is discussed.(ABSTRACT TRUNCATED AT 400 WORDS)

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