Life on the Salvage Path: The Deoxynucleoside Kinases of Lactobacillus acidophilus R-26

摘要: In Lactobacillus acidophilus R-26, the synthesis of DNA precursor deoxynucleotides occurs exclusively by salvage deoxynucleosides, beginning with phosphorylation four deoxynucleoside kinases. Subunits bearing three these activities are uniquely organized into two heterodimers, deoxyadenosine/deoxycytidine kinase (dAK/dCK) and deoxyadenosine/deoxyguanosine (dAK/dGK), which, along a distinct deoxythymidine (TK), catalyze parallel first committed steps dNTP biosynthesis. Whereas TK is common to most prokaryotes (and eukaryotes), other that emphasis this review quite unusual in bacteria. Each activity regulated cis its homologous end-product (dNTP) which understood act as multisubstrate inhibitor capable binding both nucleoside phosphate subsites. Conversely, inactive dAK subunit progressively activated 1) association dGK or dCK 2) conformationally driven heterotropic affect dGuo dCyd bound opposing subunit. Limited proteolysis has proven be powerful probe conformational states. Further indication structural differences between (or dCK) former follows an ordered kinetic path, while exhibits rapid-equilibrium random kinetics. The multi-substrate behavior provides convenient new diagnostic tool for distinguishing mechanisms. Tandem dak-dgk genes have been cloned from expressed Escherichia coli dAK/dGK, utilizing associated promoter. Sequence alignments reveal 65% identity their 61% derived amino acid sequences. Encoded N-terminal sequences identical 18 residues, subunits share conserved adenylate viral TK. A more element, appears play role activation dAK, resembles G2 loop p21ras. Remarkably, no gene(s) dAK/dCK pair could found. Comparisons sequences, isoelectric pHs masses strongly indicated native sequence, except at extreme N-termini (M–IVL –TVTVL dGK), suggesting processing . Accordingly, deletion codons 2 3 dgk resulted expression E host; properties indistinguishable those dAK/dCK. Subcloning engineered dck gene active homodimers, each virtually unchanged Km toward primary deoxynucleoside. However, human dCK, dGK) homodimer secondary much larger Kms towards dAdo dCyd). dCTP dGTP) best all respective homodimer. Fully heterodimers can reconstituted simply mixing independently (inactive) dAK. © 1998 Academic Press