Unusual Cofactor Research Articles

Cloning and Characterization of a Human STE20-like Protein Kinase with Unusual Cofactor Requirements

We cloned and characterized a novel human member of the STE20 serine/threonine protein kinase family named mst-3. Based on its domain structure, mst-3 belongs to the SPS1 subgroup of STE20-like proteins, which includes germinal center (GC) kinase, hematopoietic progenitor kinase (HPK), kinase homologous to STE20/SPS-1 (KHS), kinases responsive to stress (KRS1/2), the mammalian STE20-like kinases (mst1/2), and the recently published STE20/oxidant stress response kinase SOK-1. mst-3 is most closely related to SOK-1, with 88% amino acid similarity in the kinase domain. The similarity of the mst-3 kinase domain to STE20 is 42%. The mst-3 transcript is ubiquitously expressed, and the protein was found in all human, mouse, and monkey cell lines tested. An in vitro kinase assay showed that mst-3 can phosphorylate basic exogenous substrates as well as itself. Interestingly, mst-3 prefers Mn2+ to Mg2+ as a divalent cation and can use both GTP and ATP as phosphate donors. Like SOK-1, mst-3 is activated by autophosphorylation. However, a physiological stimulus of mst-3 activity was not identified. mst-3 activity does not change upon exposure to several mitogenic and stress stimuli. Overexpression of mst-3 wild-type or kinase dead protein affects neither the extracellular signal-regulated kinases (ERK1/2 or ERK6), c-Jun N-terminal kinase (JNK), p38, nor pp70S6 kinase, suggesting that mst-3 is part of a novel signaling pathway.

Two amine oxidases from Aspergillus niger AKU 3302 contain topa quinone as the cofactor: unusual cofactor link to the glutamyl residue occurs only at one of the enzymes

Amine oxidases (EC 1.4.3.6) from Aspergillus niger, AO-I (2 × 75 kDa) and AO-II (80 kDa), were examined to determine the cofactor structure. Inactivated with p-nitrophenylhydrazine, they showed absorption and fluorescence spectra similar to those published for other copper amine oxidases and to topa hydantoin p-nitrophenylhydrazone. After digestion by thermolysin and pronase, cofactor peptides were purified by HPLC and sequenced. For thermolytic peptides, a typical topa consensus sequence, Asn-X-Glu-Tyr, was obtained for AO-II, although in case of AO-I it overlapped with Val-Val-Ile-Glu-Pro-Tyr-Gly. For pronase peptides of AO-I, only the latter sequence was obtained. NMR and mass spectroscopy confirmed the residue X as topa p-nitrophenylhydrazone in AO-II and revealed the presence of a residue Z attached to the Glu in the peptide Val-Val-Ile-Glu( Z)-Pro of AO-I. This residue was separated from the peptide by hydrolysis and identified as a product derived from topa quinone. The data, together with amino-acid sequence of AO-I, confer strong evidence for topa quinone as the cofactor, bound in the typical consensus sequence. Raman spectra of the p-nitrophenyl-hydrazone derivative of AO-I and its pronase peptide showed essentially the same peaks matching to a model compound for topa p-nitrophenylhydrazone. However, there may exist an unusual ester link between the topa-404 and Glu-145 in the native enzyme.

Naphthalene degradation via salicylate and gentisate by Rhodococcus sp. strain B4.

Rhodococcus sp. strain B4, isolated from a soil sample contaminated with polycyclic aromatic hydrocarbons, grows with naphthalene as the sole source of carbon and energy. Salicylate and gentisate were identified as intermediates in the catabolism of naphthalene. In contrast to the well-studied catabolic pathway encoded by the NAH7 plasmid of Pseudomonas putida, salicylate does not induce the genes of the naphthalene-degradative pathway in Rhodococcus sp. strain B4. The key enzymes of naphthalene degradation in Rhodococcus sp. strain B4 have unusual cofactor requirements. The 1,2-dihydroxynaphthalene oxygenase activity depends on NADH and the salicylate 5-hydroxylase requires NADPH, ATP, and coenzyme A.

Structure and Mechanism of Citrate Synthase

Publisher Summary This chapter discusses the structure and mechanism of citrate synthase. The enzyme citrate synthase that catalyzes the reaction at the entry to the citric acid cycle, forms an outstanding system for investigation of enzymatic mechanisms, enzyme–substrate interactions, conformational change, protein folding, quaternary assembly of biological structures, and organelle targeting. Citrate synthase provides a system in which the simplest example of quaternary structural assembly to an active oligomer can be studied. In eukaryotes, the enzyme is a dimer in which each subunit contributes functional groups to the active site of the other. The dimer can be reversibly denatured to inactive monomers. The enzyme requires no unusual cofactors or metal ions for activity; it is one of few enzymes that can catalyze the synthesis of a carbon–carbon bond, and it does so at a rate that approaches the diffusion limit. Citrate synthase has been discovered in essentially every organism examined. The enzyme catalyzes the reversible condensation of acetyl-CoA to form citryl-CoA, which is subsequently hydrolyzed to yield citrate and coenzyme A. Thus, the enzyme closes the loop of the citric acid cycle, regenerating the six-carbon compound citrate from the four-carbon compound oxaloacetate.

Practical Processing with Cofactor -Requiring Enzymes

Abstract The use of cofactor requiring enzymes in practical processes is dependent on regeneration and multiple reuse of the cofactor. Various methods currently available for the accomplishment of this goal are evaluated. We conclude that, in most instances, enzymatic regeneration techniques are superior to chemical regeneration techniques. We also conclude that, except in very unusual cases, cofactor regeneration and reuse is most efficient when processing enzymes as well as cofactor regenerating enzymes are contained in the same reactor. A cofactor classification based on regeneration requirements is proposed. Methods for the retention of both modified (for example, macro molecular) and native cofactors within the process reactor are discussed, as well as various cofactor recovery techniques. Finally, a prototype enzyme reactor employing native cofactor without retention, and allowing high substrate to product conversion with an extremely high number of cofactor cycles, is proposed. Utilizing this react...