A broader active site in Pyrococcus horikoshii CoA disulfide reductase accommodates larger substrates and reveals evidence of subunit asymmetry

Abstract

Within the family of pyridine nucleotide disulfide oxidoreductase (PNDOR), enzymes are a group of single‐cysteine containing FAD‐dependent reductases that utilize a tightly bound coenzyme A to assist in the NAD(P)H‐dependent reduction of di‐, per‐, and polysulfide substrates in bacteria and archaea. For many of these homodimeric enzymes, it has proved difficult to determine the substrate specificity and metabolic function based on sequence and genome analysis alone. Coenzyme A‐disulfide reductase (CoADR) isolated from Pyrococcus horikoshii ( ph CoADR) reduces Co‐A per‐ and polysulfides, but, unlike other highly homologous members of this group, is a poor CoA disulfide reductase. The ph CoADR structure has a narrower access channel for CoA substrates, which suggested that this restriction might be responsible for the enzyme's poor activity toward the bulky CoA disulfide substrate. To test this hypothesis, the substrate channel was widened by making four mutations along the channel wall (Y65A, Y66A, P67G, and H367G). The structure of the quadruple mutant shows a widened substrate channel, which is supported by a fourfold increase in k cat for the NAD(P)H‐dependent reduction of CoA disulfide and enhanced activity toward the substrate at lower temperatures. Anaerobic titrations of the enzyme with NADH revealed a half‐site reactivity not observed with the wild‐type enzyme in which one subunit of the enzyme could be fully reduced to an EH 4 state, while the other remained in an EH 2 or EH 2 ·NADH state. These results suggest that for these closely related enzymes, substrate channel morphology is an important determinant of substrate specificity, and homology modeling will be the preferred technique for predicting function among PNDORs.

Thioredoxin reductase from Bacillus cereus exhibits distinct reduction and NADPH‐binding properties

“…It can, however, not be ruled out that the pure half‐of‐the‐site reactivity is a crystallographically induced effect of a possible weak negative cooperativity in solution or a preexisting asymmetry, thereby not exhibiting cooperativity [ 53 , 54 ]. Nevertheless, half‐site reactivity or subunit asymmetry is observed in several other pyridine nucleotide‐disulfide oxidoreductases [ 55 ]. The two conformational states of TrxR, FO and FR, are interchanged by the ˜ 67° rotation and could also lead to alternating NADPH binding and catalysis within the TrxR dimer, functioning in a sequential mode of action.…”

“…Nevertheless, half-site reactivity or subunit asymmetry is observed in several other pyridine nucleotide-disulfide oxidoreductases [55]. The two conformational states of TrxR, FO and FR, are interchanged by the ~67º rotation, and could also lead to alternating NADPH binding and catalysis within the TrxR dimer, functioning in a sequential mode of action.…”

New Insights into the Cooperativity and Dynamics of Dimeric Enzymes

“…Many studies have noted that certain enzymes can enhance the rate of typical chemical reactions by more than 10 17 times . Determining substrate specificity and metabolic function based solely on sequence and genomic analysis is challenging for many homodimeric enzymes . Consequently, a range of precise biochemical techniques have been developed and enhanced to provide greater insight into enzymatic processes.…”

“…118 Determining substrate specificity and metabolic function based solely on sequence and genomic analysis is challenging for many homodimeric enzymes. 119 Consequently, a range of precise biochemical techniques have been developed and enhanced to provide greater insight into enzymatic processes. These methodologies encompass various biophysical experimental techniques, including X-ray crystallography, 120 NMR, 121,122 mass spectrometry (MS) detection, 123 CD, 124 ITC, 125,126 temperature-jump, 127 and stopped-flow analysis, 128 among others.…”

Structural and Kinetic Characterization of Hyperthermophilic NADH-Dependent Persulfide Reductase from Archaeoglobus fulgidus

“…These observations suggest disulfides have restricted access to the AfNpsr active site. CoADR from Pyrococcus horikoshii, which has 40% sequence identity and 56% similarity to AfNpsr, has similar substrate preference toward per-and polysulfides over disulfides [12].…”