SalM is a short-chain dehydrogenase/reductase enzyme from the marine actinomycete Salinispora tropica that is involved in the biosynthesis of chloroethylmalonyl-CoA, a novel halogenated polyketide synthase extender unit of the proteasome inhibitor salinosporamide A. SalM was heterologously overexpressed in Escherichia coli and characterized in vitro for its substrate specificity, kinetics, and reaction profile. A sensitive real-time (13)C NMR assay was developed to visualize the oxidation of 5-chloro-5-deoxy-D-ribose to 5-chloro-5-deoxy-D-ribono-γ-lactone in an NAD(+)-dependent reaction, followed by spontaneous lactone hydrolysis to 5-chloro-5-deoxy-D-ribonate. Although short-chain dehydrogenase/reductase enzymes are widely regarded as metal-independent, a strong divalent metal cation dependence for Mg(2+), Ca(2+), or Mn(2+) was observed with SalM. Oxidative activity was also measured with the alternative substrates D-erythrose and D-ribose, making SalM the first reported stereospecific non-phosphorylative ribose 1-dehydrogenase.