Role of Lysine 39 of Alanine Racemase from Bacillus stearothermophilus That Binds Pyridoxal 5′-Phosphate CHEMICAL RESCUE STUDIES OF Lys39 → Ala MUTANT

We have studied the stereospecificities of various pyridoxal 5′-phosphate dependent enzymes for the hydrogen transfer between the C-4′ of a bound coenzyme and the C-2 of a substrate in the transamination catalyzed by the enzymes. Prior to our studies, pyridoxal enzymes so far studied were reported to catalyze the hydrogen transfer only on the si-face of the planar imine intermediate formed from substrate and coenzyme. This finding had been considered as the evidence that pyridoxal enzymes have evolved divergently from a common ancestral protein, because identity in the stereospecificity reflects the similarity in the active-site structure, in particular in the geometrical relationship between the coenzyme and the active site base participating in the hydrogen transfer. However, we found that d-amino acid aminotransferase, branched-chain l-amino acid aminotransferase, and 4-amino-4-deoxychorismate lyase catalyze the re-face specific hydrogen transfer, and that amino acid racemases catalyze the nonstereospecific hydrogen transfer. These findings suggest the convergent evolution of pyridoxal enzymes. Crystallographical studies have shown that the stereospecificity reflects the active-site structure of the enzymes, and that the enzymes with the same fold exhibit the same stereospecificity. The active site structure with the catalytic base being situated on the specific face of the cofactor has been conserved during the evolution among the pyridoxal enzymes of the same family. © 2001 John Wiley & Sons, Inc. and The Japan Chemical Journal Forum Chem Rec 1:373–384, 2001

Stereospecificity for the hydrogen transfer of pyridoxal enzyme reactions

The maltose binding protein (MBP or MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides. It is synthesized in the cytoplasm with an N-terminal signal peptide that is cleaved upon export. We examined whether active MBP could assemble into an active protein in bacteria, from N- and COOH-terminal complementary protein fragments encoded by distinct, engineered segments of its structural gene. We found export and functional periplasmic assembly of MBP fragments, despite the complex polypeptide chain topology of this protein, if two conditions were satisfied. First, each of the two fragments must carry a signal peptide. Second, the boundaries between the two fragments must correspond to a permissive site within the protein. Functional assembly of active MBP occurred in five cases where these conditions were met: sites after residues 133, 161, 206, 285 and 303; but not in three other cases where the break junction corresponded to a non-permissive site: after residues 31, 120 and 339. Thus, permissive sites which were initially characterized because they could accept extensive genetic insertion/deletion modifications without loss of most biological properties provide a means of defining complementing protein fragments. This observation opens a way to study genetically the relationships between protein export and folding into the periplasm.

In vivo assembly of active maltose binding protein from independently exported protein fragments.

Expression of glr (murI, dga) gene encoding glutamate racemase in Escherichia coli.

https://escholarship.org/content/qt0832b0bq/qt0832b0bq.pdf?t=oemkm6

Role of pyridoxal 5'-phosphate in the structural stabilization of O-acetylserine sulfhydrylase.

Thermostable alanine racemase of Bacillus stearothermophilus. Construction and expression of active fragmentary enzyme.

The guanidine hydrochloride-induced subunit dissociation and unfolding of thermostable alanine racemase from Bacillus stearothermophilus have been studied by circular dichroism, fluorescence and absorption spectroscopies, and gel filtration. The overall process was found to be reversible: more than 75% of the original activity was recovered upon reduction of the denaturant concentration. In the range of 0.6 to 1.5 M guanidine hydrochloride, the dimeric enzyme was dissociated into a monomeric form, which was catalytically inactive. The monomeric enzyme appeared to bind the cofactor pyridoxal phosphate by a non-covalent linkage, although the native dimeric enzyme binds the cofactor through an aldimine Schiff base linkage. The monomer was mostly unfolded, with the transition occurring in the range of 1.8 to 2.2 M guanidine hydrochloride.

Thermostable alanine racemase of Bacillus stearothermophilus: subunit dissociation and unfolding.

Thermostable Alanine Racemase of Bacillus Stearothermophilus: Interdependent Folding of Two Domains into an Active Complex

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Thermostable alanine racemase of Bacillus stearothermophilus. Construction and expression of active fragmentary enzyme.

Thermostable alanine racemase of Bacillus stearothermophilus: subunit dissociation and unfolding.

Thermostable Alanine Racemase of Bacillus Stearothermophilus: Interdependent Folding of Two Domains into an Active Complex