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Isoleucine Biosynthesis in Leptospira interrogans Serotype lai Strain 56601 Proceeds via a Threonine-Independent Pathway ,. Hai Xu, 2004.Three leuA-like protein-coding sequences were identified in Leptospira interrogans . One of these, the cimA gene, was shown to encode citramalate synthase (EC 4.1.3.-) . The other two encoded  -isopropylmalate
synthase (EC 4.1.3.12) . Expressed in Escherichia coli, the
citramalate synthase was purified and characterized . Although its
activity was relatively low, it was strictly specific for pyruvate as
the keto acid substrate . Unlike the citramalate synthase of the
thermophile Methanococcus jannaschii, the L . interrogans
enzyme is temperature sensitive but exhibits a much lower Km
(0.04 mM) for pyruvate . The reaction product was characterized as (R)-citramalate,
and the proposed ß-methyl-D-malate pathway was
further confirmed by demonstrating that citraconate was the substrate
for the following reaction . This alternative pathway for isoleucine
biosynthesis from pyruvate was analyzed both in vitro by assays of
leptospiral isopropylmalate isomerase (EC 4.2.1.33) and
ß-isopropylmalate dehydrogenase (EC 1.1.1.85) in E . coli
extracts bearing the corresponding clones and in vivo by
complementation of E . coli ilvA, leuC/D, and
leuB mutants . Thus, the existence of a leucine-like pathway for
isoleucine biosynthesis in L . interrogans under physiological
conditions was unequivocally proven . Significant variations in either
the enzymatic activities or mRNA levels of the cimA and
leuA genes were detected in L . interrogans grown on minimal
medium supplemented with different levels of the corresponding
amino acids or in cells grown on serum-containing rich medium . The
similarity of this metabolic pathway in leptospires and archaea is
consistent with the evolutionarily primitive status of the
eubacterial spirochetes .
Moritella Cold-Active Dihydrofolate Reductase: Are There Natural Limits to Optimization of Catalytic Efficiency at Low Temperature?. Ying Xu, 2003.Adapting metabolic enzymes of microorganisms to low temperature environments may require a difficult compromise between velocity and affinity . We have investigated catalytic efficiency in a key metabolic enzyme (dihydrofolate reductase) of Moritella profunda sp . nov., a strictly psychrophilic bacterium with a maximal growth rate at 2°C or less . The enzyme is monomeric (Mr = 18,291), 55% identical to its Escherichia coli counterpart, and displays Tm and denaturation enthalpy changes much lower than E . coli and Thermotoga maritima homologues . Its stability curve indicates a maximum stability above the temperature range of the organism, and predicts cold denaturation below 0°C . At mesophilic temperatures the apparent Km value for dihydrofolate is 50- to 80-fold higher than for E . coli, Lactobacillus casei, and T . maritima dihydrofolate reductases, whereas the apparent Km value for NADPH, though higher, remains in the same order of magnitude . At 5°C these values are not significantly modified . The enzyme is also much less sensitive than its E . coli counterpart to the inhibitors methotrexate and trimethoprim . The catalytic efficiency (kcat/Km) with respect to dihydrofolate is thus much lower than in the other three bacteria . The higher affinity for NADPH could have been maintained by selection since NADPH assists the release of the product tetrahydrofolate . Dihydrofolate reductase adaptation to low temperature thus appears to have entailed a pronounced trade-off between affinity and catalytic velocity . The kinetic features of this psychrophilic protein suggest that enzyme adaptation to low temperature may be constrained by natural limits to optimization of catalytic efficiency . Purification and Characterization of Brochocin A and Brochocin B(10-43), a Functional Fragment Generated by Heterologous Expression in Carnobacterium piscicola. Sylvie Garneau, 2003.Brochothrix campestris ATCC 43754 produces a heat-stable, two-component, nonlantibiotic, class IIb bacteriocin, brochocin C (BrcC), that is active against a broad range of gram-positive bacteria, including spores of Clostridium botulinum . An improved purification method was developed for BrcC, in which n-butanol and chloroform extraction are used . Mass spectral characterization of the two components, brochocin A (BrcA) and brochocin B (BrcB), showed that both components are excreted into the medium by B . campestris as mature peptides consisting of 59 and 43 amino acids, respectively . Separate expression clones of BrcA and BrcB were constructed previously in Carnobacterium piscicola LV17C, but the products were not chemically characterized . Purification by the new protocol showed that BrcA is expressed as the mature 59-amino-acid peptide but that BrcB is produced by C . piscicola as a fragment, BrcB(10-43), which is cleaved at an internal Gly-Gly site . This fragment is not antimicrobial by itself, but in combination with BrcA it displays the full activity of the BrcC complex . Circular dichroism measurements revealed a high ß-sheet content in the secondary structure of both BrcA and BrcB(10-43), as well as in a 1:1 BrcA-BrcB(10-43) mixture . Separate expression clones of brcA and brcB were also constructed in Escherichia coli, but these clones only produced multiple fragments of the desired peptides with little or no activity .
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