Gayle Burns

Oxidation of glycine by Pseudomonas putida requires a specific lipoamide dehydrogenase

Pseudomonas putida produces two lipoamide dehydrogenases with molecular weights of 49,000 and 56,000 designated LPD-val and LPD-glc, respectively. LPD-val is required for oxidation of valine, since it is specifically utilized as the E3 component of branched-chain keto acid dehydrogenase. Since glycine oxidation by bacteria and mammals also requires lipoamide dehydrogenase, we desired to determine which lipoamide dehydrogenase would be used by the P. putida glycine oxidation system. When grown in a medium with glycine as the sole nitrogen source, P. putida produced a single lipoamide dehydrogenase with a molecular weight of 56,000 and which reacted with antiserum to LPD-glc. The partially purified glycine oxidation system from P. putida was stimulated by LPD-glc but not by LPD-val and was inhibited by anti-LPD-glc, but not by anti-LPD-val. It was not possible to detect LPD-val in extracts of cells grown in glucose-glycine medium by the use of anti-LPD-val. LPD-glc was five times as active as LPD-val in catalyzing the oxidation of purified protein H, the heat-stable, lipoic acid-containing protein of the glycine oxidation system. These results indicate that LPD-glc is specifically utilized for glycine oxidation in P. putida.

Relationship of lipoamide dehydrogenases from Pseudomonas putida to other FAD-linked dehydrogenases.

Pseudomonas putida produces two lipoamide dehydrogenases, LPD‐glc and LPD‐val. LPD‐val is specifically required as the lipoamide dehydrogenase of branched‐chain keto acid dehydrogenase and LPD‐glc fulfills all other requirements for lipoamide dehydrogenase. Both proteins are dimers with one FAD per subunit. LPD‐glc has an absorption maximum at 455 nm, but LPD‐val has a maximum at 460 nm. Comparison of amino acid compositions revealed that LPD‐glc was more closely related to Escherichia coli and pig heart lipoamide dehydrogenase than to LPD‐val. LPD‐val did not appear to be closely related to any of the proteins compared with the possible exception of mercuric reductase.

Construction of chromosomal recA mutants of Pseudomonas putida PpG2.

The recA gene of Pseudomonas putida PpG2 was cloned by complementation of the recA mutations of Escherichia coli strains DH5 alpha and HB101. The nucleotide sequence of the DNA fragment was determined and shown to contain recA and a downstream partial open reading frame. Two mutants of P. putida PpG2, strains JS387 and JS388, were constructed by insertional inactivation of recA with a tetracycline-resistance gene in both orientations. Both mutants acquired sensitivity to methyl methanesulfonate (MMS) and both failed to undergo homologous recombination. While the recA mutation of P. putida JS388 was complemented in trans by recA of P. putida, the JS387 mutant was difficult to transform and transformants exhibited varying degrees of sensitivity to MMS. Therefore, P. putida JS388 can be used as a carrier of recombinant plasmids, but JS387 is not a suitable host for this purpose.